SPECIATION AND ANTIBIOGRAM PATTERN OF TRIBE PROTEEAE WITH SPECIAL REFERENCE TO EXTENDED SPECTRUM BETA LACTAMASE (ESBL) AND CARBAPENEMASE DETECTION

Background:Proteusis a common uropathogen causing urinary tract infections in catheterised patients and those with urinary tract abnormalities. It may also lead to pyelonephritis, renal stones & bacteraemia. Multidrug resistant Proteeae isolates are major problem in treating nosocomial infections. Beta lactamase production is being increasingly demonstrated in most of the Proteus species. Apart from ESBL, Carbapenemase production is also emerging, thereby limiting the treatment options. Aim: To isolate, speciate and study the antibiotic resistance pattern of the Proteeae isolates. To identify the extended spectrum beta lactamase andcarbapenemase producing strains of Proteeae isolates by employing phenotypic methods. Materials and Methods: A total of 145 isolates of Proteus species isolated from different clinical samples- urine, pus and sputum, were included in this study. Antibiotic sensitivity testing was performed by Kirby-Bauer disk diffusion method. Screening tests for ESBL &Carbapenemase production was confirmed by Disk diffusion method and Modified Hodge test respectively. Results: Out of the 145 Proteeae isolates, from various clinical samples 70 were from wound swabs, 62 from urine, and 13 from respiratory specimens. The species were identified as 64 P.mirabilis, 48 P.vulgaris, 19 M.morganii, 5 Prov.stuartii and 9 Prov.rettgeri.Different antibiotic resistance patterns were observed in different species. Providencia species showed resistance to most of the antibiotics than the Proteus species and M morganii. Of the total, 52 (36%) were ESBL producers. Among the ESBL producers 6 (11.5%) were Carbapenamase producers. Conclusion: The increasing incidence of multi drug resistant strains in Tribe Proteeae has made antimicrobial susceptibility testing more important. Avoidance of indiscriminate use of antibiotics is the first step in prevention of newly emerging drug resistant strains.

Phenotypic identification & molecular detection of blandm-1 gene in multidrug resistant Gram-negative bacilli in a tertiary care centre.

Background & objectives: Carbapenemase-producing Enterobacteriaceae isolates have been increasingly identified worldwide. Though molecular data regarding New Delhi metallo-beta-lactamase-1 (NDM-1) producers are available, data regarding their rate of infection in a hospital setting and percentage among different clinical isolates are scarce. Hence, this study was undertaken to determine the occurrence of blaNDM-1 gene among clinical isolates of multidrug resistant Gram-negative bacilli (MDRGNB) in a tertiary care centre in Bangalore, Karnataka, India. Methods: A total of 74 MDRGNB isolates were studied. These were screened for MBL production by phenotypic assays such as double disk synergy test (DDST) and Modified Hodge’s test (MHT). PCR was performed for the molecular detection of the gene and antibiograms were confirmed by automated bacteriology system. Results: Of the 74 MDRGNB isolates, 34 were positive for blaNDM-1 gene. All isolates were resistant to aztreonam and two isolates were resistant to tigecycline. Complete resistance to the tested carbapenems was seen in 28 (82.35%) of the positive isolates whereas variable carbapenem resistance was seen in six (17.64%) of the positive clinical isolates. Of the total 34 PCR positive isolates, 33 (97.05%) NDM-1 producers were identified by DDST and 26 (76.47%) by MHT as producers of MBL. Interpretation & conclusions: A high percentage of plasmid encoded NDM was noted in MDRGNB. Phenotypic and molecular screening should be employed along with routine antimicrobial susceptibility testing to reflect the true number of metallo-beta-lactamase producers.

Application analysis of double-disk synergy test in AmpC β-lactamase detection / 国际检验医学杂志

Objective To investigate and analyze the double-disk inhibiting synergy test for detecting AmpC β-lactamase pro-duced by Klebsiella pneumoniae and to evaluate its application value in clinical laboratory.Methods The cefoxitin disk agar diffu-sion method,cefoxitin three-dimensional method,double-disk inhibiting synergy test and drug resistance gene multiplex PCR assay were adopted to detect the clinically isolated bacterial strains.Results Among 137 clinically isolated strains of Klebsiella pneumoni-ae,22 strains were insensitive to cefoxitin and 11 strains were positive by the three-dimensional method;in the double-disk inhibiting synergy test,18 strains were positive for the FOX/FOX+PBA group and 11 strains were positive for the CTT/CTT+PBA group respectively;in the multiplex PCR assay,19 strains were positive.The coincidence rate of the cefoxitin three-dimensional method and multiplex PCR methods was 47.4%(9/19),in the double-disk inhibiting synergy test,the coincidence rate of the positive re-sults in the CTT/CTT+PBA group and the multiplex PCR methods was 57.9%(11/19);the coincidence rate of the FOX/FOX+PBA group and multiplex PCR methods was 94.7%(18/19).Conclusion The double-disk inhibiting synergy test is simple with highly accurate results,in which the FOX/FOX+PBA double-disk synergy test could be applied to detect AmpC β-lactamase pro-duced by clinical isolates of Klebsiella pneumoniae.

Comparison of MicroScan and Phoenix Automated Systems for Detection of Extended-Spectrum beta-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae

The aim of this study was to evaluate the performances of MicroScan (Siemens Healthcare, USA) and Phoenix (Becton Dickinson Diagnostic Systems, USA) automated systems for the detection of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae. ESBL-producers were detected from 18 E. coli strains and 26 K. pneumoniae strains using MicroScan, Phoenix, and double-disk synergy test (DDST). The ESBL types were determined by PCR direct sequencing. ESBL genes were detected in 38 (86.4%) of the 44 test strains. The sensitivities of MicroScan, Phoenix, and DDST were 94.6%, 79%, and 89.5%, respectively. Both MicroScan and Phoenix provided acceptable results for the examination of clinical isolates.

Moleculoepidemiological Characteristics of Extended-Spectrum beta-Lactamase Producing Escherichia coli and Klebsiella pneumoniae Strains in Daegu / 대한진단검사의학회지

BACKGROUND: The importance of extended-spectrum beta-lactamases (ESBL) produced in gramnegative bacilli is now well recognized, but most clinical laboratories have problems in detecting and interpreting ESBL and implicating the findings in nosocomial infections caused by ESBL producing gram-negative bacilli. The present study aims primarily to evaluate the distributions of these enzymes among Escherichia coli and Klebsiella pneumoniae, the most frequent isolates of Enterobacteriaceae producing ESBL, to differentiate the types of enzymes in theses isolates and finally to relate the clonality of specific types within a part of Daegu city. METHODS: The clinical isolates of 1, 242 E. coli and 859 K. pneumoniae were screened for ESBL production by the disk diffusion method of the National Committee of Clinical Laboratory Standard, and it was confirmed by the double-disk synergy test (DDS). Antimicrobial susceptibility test was performed by the agar dilution method. The presence of -lactamase was tested by polymerase chain reaction (PCR) and plasmid analysis. Isoelectric focusing and nucleotide sequence analysis were performed to evaluate ESBL types. Pulsed-field gel electrophoresis (PFGE) of XbaI-digested DNA fragments was carried out to determine the extend of clonality within the hospital. RESULTS: Of 34 isolates of E. coli and 31 isolates of K. pneumoniae ramdomly selected from those isolates screened for ESBL production were further tested by DDS to confirm its production: 30 (88.2%) E. coli and 29 (93.5%) K. pneumoniae were positive. TEM-52 and SHV-12 were present both in E. coli and K. pneumoniae, but SHV-2a was distributed only in K. pneumoniae. The resistance was transferable in 66.7% of E. coli and 68.9% of K. pneumoniae. Six and 5 PFGE patterns were shown by E. coli and K. pneumoniae, respectively. Among the 5 patterns of K. pneumoniae, type B was dominant, suggesting a clonal outbreak in the hospital. CONCLUSIONS: The ESBL specific enzyme types were TEM-52, SHV-2a and SHV-12. Despite many different PFGE patterns of the ESBL producing isolates, a few outbreak and edemic clones appear to be prevalent in Dongsan Medical Center.

Comparison of Vitek ESBL Test and Other Methods for Detecting Extended-Spectrum -Lactamase-Producing Escherichia coli and Klebsiella Species / 대한임상병리학회지

BACKGROUND: Because extended-spectrum -lactamase (ESBL) producing strains can frequent-ly cause therapeutic failure and infectious outbreaks in hospitals, rapid and accurate detection of these strains are important. We compared the Vitek ESBL test with the NCCLS ESBL phenotypic confirmatory test by disk diffusion (NCCLS ESBL test) and double disk synergy test (DDST). METHODS: For a total of 316 clinical isolates composed of Escherichia coli (184), Klebsiella pneu-moniae (120) and Klebsiella oxytoca (12), we performed the Vitek ESBL test and the NCCLS ESBL test. For sixty-eight ESBL producing isolates, the Vitek ESBL test was compared with the NCCLS ESBL test and the DDST. The ESBL producer was defined as an organism showing an increase in the inhibited zone diameter of >or=5 mm for either cefotaxime or ceftazidime in combination with clavu-lanic acid versus its single test. The DDST was performed with 20 mm and 30 mm for interdisk diam-eter. For seven false negative isolates in the Vitek ESBL test, the DDST of cefepime was performed. RESULTS: Compared with the NCCLS ESBL test, the Vitek ESBL test showed one false positive (specificity, 99.6%), seven false negatives (sensitivity, 89.7%) and 97.5% agreement. Seven false negative isolates of the Vitek ESBL test were the cefoxitin-resistant ESBL producer. In positivity for the NCCLS ESBL test of 68 ESBL producing isolates, cefotaxime-clavulanic acid and ceftazidime-clavulanic acid were 94% and 91%. Cefotaxime, ceftazidime, aztreonam and ceftriaxone showed 95/90%, 100/55%, 100/85% and 95/80% positivity in double-disk synergy with amoxicillin-clavulanic acid (AMC) for 20/30 mm of the interdisk diameter respectively. For seven false negative isolates of the Vitek ESBL test, cefepime showed a distinct synergic effect with AMC. CONCLUSIONS: The Vitek ESBL test may be a useful method for clinical laboratories due to its easy, rapid and sensitive method but its method was less sensitive to cefoxitin-resistant ESBL. For these cases, if the NCCLS ESBL test or DDST with cefepime are added, the detection rate of the ESBL pro-ducer can be augmented.

Etest as a Method of Detecting Extended-Spectrum beta-Lactamase / 감염

BACKGROUND: Detection of extended-spectrum beta-lactamase (ESBL) expression is difficult in ordinary clinical laboratories. The Etest has been introduced into clinical settings for the rapid identification of ESBL. The principle behind the Etest is to compare the minimal inhibitory concentration (MIC) of ceftazidime alone with the MIC of ceftazidime with clavulanic acid. The aim of this study was to evaluate the efficiency of the Etest for the detection of ESBL in Korea, where antimicrobial resistance rates are high. METHODS: The double disk synergy test and the Etest were performed simultaneously. The results of the clinical isolates were compared to those of strains producing TEM-1, TEM-2, and SHV-1 as negative controls. The results of the double disk synergy test and the E-test were confirmed by isoelectric focusing of beta-lactamase extracted from suspicious ESBL-producing strains. RESULTS: MIC determination using the standard agar dilution method according to the National Committee for Clinical Laboratory Standards revealed that a total of 48 strains were resistant or intermediate against one or more antibiotics of the third generation cephalosporins. These strains included five strains of E. coli, 14 of S. marcescens, seven of K. pneumoniae, 18 of Enterobacter spp., and four of Citrobacter spp. Sixteen (33%) of the strains, including five strains of E. coli, three of S. marcescens, five of K. pneumoniae, and three of Enterobacter spp. were ESBL- producing strains that were confirmed by double disk synergy test. Thirteen (81%) of the strains of ESBL- producing organisms were detected by Etest, but the remaining three strains (19%) were undetectable by Etest alone. CONCLUSION: The accuracy of Etest for the detection of ESBL was not high, but the efficiency of Etest as the primary screening method of a large number of clinical isolates was appreciable regarding efficiency and rapidity.

Etest as a Method of Detecting Extended-Spectrum beta-Lactamase / 감염

BACKGROUND: Detection of extended-spectrum beta-lactamase (ESBL) expression is difficult in ordinary clinical laboratories. The Etest has been introduced into clinical settings for the rapid identification of ESBL. The principle behind the Etest is to compare the minimal inhibitory concentration (MIC) of ceftazidime alone with the MIC of ceftazidime with clavulanic acid. The aim of this study was to evaluate the efficiency of the Etest for the detection of ESBL in Korea, where antimicrobial resistance rates are high. METHODS: The double disk synergy test and the Etest were performed simultaneously. The results of the clinical isolates were compared to those of strains producing TEM-1, TEM-2, and SHV-1 as negative controls. The results of the double disk synergy test and the E-test were confirmed by isoelectric focusing of beta-lactamase extracted from suspicious ESBL-producing strains. RESULTS: MIC determination using the standard agar dilution method according to the National Committee for Clinical Laboratory Standards revealed that a total of 48 strains were resistant or intermediate against one or more antibiotics of the third generation cephalosporins. These strains included five strains of E. coli, 14 of S. marcescens, seven of K. pneumoniae, 18 of Enterobacter spp., and four of Citrobacter spp. Sixteen (33%) of the strains, including five strains of E. coli, three of S. marcescens, five of K. pneumoniae, and three of Enterobacter spp. were ESBL- producing strains that were confirmed by double disk synergy test. Thirteen (81%) of the strains of ESBL- producing organisms were detected by Etest, but the remaining three strains (19%) were undetectable by Etest alone. CONCLUSION: The accuracy of Etest for the detection of ESBL was not high, but the efficiency of Etest as the primary screening method of a large number of clinical isolates was appreciable regarding efficiency and rapidity.

Evaluation of the Method to Screen Isolates of Extended-Spectrum -Lactamase-Producing Klebsiella pneumoniae and Escherichia coli Using Cefpodoxime Disk / 대한임상병리학회지

BACKGROUND: The prevalence of extended-spectrum -lactamase (ESBL)-producing Klebsiella pneumoniae and Escherichia coli has been increased in Korea, but the testing and reporting ESBL-mediated resistance remains unclear. We undertook a study to evaluate the method to screen isolates of ESBL-producing K. pneumoniae and E. coli using cefpodoxime disk. METHODS: Fifty-eight strains of K. pneumoniae and 28 strains of E. coli were tested for production of ESBLs by the double disk synergy test. Susceptibility to cefpodoxime, ceftazidime, cefotaxime, and aztreonam was determined by disk diffusion method. RESULTS: All strains that produced ESBLs were resistant to cefpodoxime, whereas those that not produced ESBLs were susceptible (97%) to this agents. The disk diffusion test exhibited 100% sensitivity and 97% specificity when NCCLS conventional interpretive criteria were used. All other oxyimino- -lactam agents tested were inferior discriminators between the two groups of organisms. When NCCLS ESBL interpretive criteria were used, the disk diffusion test using cefpodoxime exhibited 100% sensitivity and 83% specificity. CONCLUSIONS: Routine disk diffusion susceptibility test with cefpodoxime disk (10g) can be used to detect strains of ESBL-producing K. pneumoniae and E. coli without include supplemental testing for ESBL production.