Enhancing Phenotypic Detection of ESBL in AmpC co-producers by using Cefepime and Tazobactam.

INTRODUCTION: Routine phenotypic methods employing clavulanate and third generation cephalosporins to detect ESBL are not promising for isolates that co-produce an inhibitor-resistant beta lactamase like AmpC. AIM: Enhancing phenotypic detection of ESBL in AmpC co-producers by using cefepime and tazobactam. MATERIALS AND METHODS: A total of 245 isolates of Escherichia coli (123), Klebsiella spp. (87), Proteus spp.(20), Enterobacter spp. (9) and Citrobacter spp.(6) obtained over a period of 2 years from January 2013 to December 2014 from urine samples of hospitalized patients were studied. The isolates were simultaneously screened for ESBL and AmpC production. AmpC production was confirmed by modified three -dimensional test (MTDT). ESBL production was confirmed by original double disc synergy test, phenotypic disc confirmatory test (PDCT) and modified double disc synergy test (MDDST) and the results compared. RESULTS: AmpC production was confirmed in 113 (46.1%) isolates by modified three dimensional test out of 143 screened positive for AmpC. Of the 192 isolates screened positive for ESBL, ESBL production was confirmed in 162 (66.1%). DDST detected ESBLs in only134 (54.7%) while additional 28 (11.4%) ESBL positive isolates were detected by MDDST. PDCT detected total 145(59.2%) ESBL positive isolates, with cefotaxime and cefotaxime + clavulanate detecting 139 (56.7%) and ceftazidime and ceftazidime + clavulanate detecting additional 6 isolates. All the 28 (11.4%) isolates which were additionally detected ESBL producers by MDDST showed positive three dimensional test i.e. AmpC co producers. DDST detected ESBL in none of AmpC positive isolates while PDCT detected ESBL in 11 isolates showing AmpC co-production. In MDDST cefepime was the best cephalosporin in detecting ESBL in presence of AmpC production. It showed synergism with amoxicillin-clavulanate in 11(39.3%) isolates and in 24(85.7%) isolates with piperacillin-tazobactam. Third generation cephalosporins -cefotaxime, ceftazidime and cefpodoxime were not able to detect ESBL in AmpC-co producers. CONCLUSION: Modification of double disc synergy tests that combine piperacillin-tazobactum with cefepime enhances the possibility of ESBL detection.

Modified Double Disc Synergy Test to Detect ESBL Production in Urinary Isolates of Escherichia coli and Klebsiella pneumoniae.

BACKGROUND AND OBJECTIVES: Various phenotypic methods are recommended in the routine practice to detect the ESBL production in gram negative bacilli. Among them, the Double Disc Synergy Test (DDST) which uses the third generation cephalosporins (3GC), is a simple and a reliable method. But the coexistence of AmpC may give false negative results. In such cases, the ESBL detection can be improved by using cefepime along with the third generation cephalosporins in DDST. METHODS: A total of 350 urinary isolates (224 Escherichia coli and 126 Klebsiella pneumoniae) were studied for ESBL production by the modified double disc test (MDDST) i.e. by using cefotaxime, ceftriaxone, cefpopdoxime (third generation cephalosporins) and cefepime ( fourth generation cephalosporin) along with a amoxicillin-clavulanate disc. RESULTS AND INTERPRETATION: ESBL production was seen in 63.4% (142/224) Escherichia coli and in 60.3% (76/126) Klebsiella pneumoniae isolates by MDDST. Among these, in twelve E.coli and five K.pneumoniae strains, only cefepime but none of the third generation cephalosporins showed synergism with amoxicillin-clavulanate. All these seventeen strains showed a clear extension of the edge of inhibition which was produced by cefepime towards the amoxicillin-clavulanate disc. These strains were further tested for AmpC co-production by the AmpC disc test and all these strains were found to be AmpC positive, thus revealing the superior activity of cefepime in detecting ESBLs in the bacteria which co-produced AmpC. A high degree of coresistance was found in the ESBL producers. CONCLUSION: The ESBL detection can be improved by MDDST by using cefepime along with the third generation cephalosporins.

Carbapenem resistance and phenotypic detection of carbapenemases in clinical isolates of Acinetobacter baumannii.

BACKGROUND AND OBJECTIVES: Multidrug-resistant Acinetobacter baumannii (MDR-Ab) reported worldwide has become one of the most difficult nosocomially acquired Gram-negative pathogens to control and treat. The clinical utility of carbapenems is under threat with the emergence of acquired carbapenemases, particularly Ambler class B metallo-lactamases (MBL). Because of the global increase in the occurrence and dissemination of MBLs, early detection is critical. This study was undertaken to detect resistance to carbapenems in clinical isolates of A. baumannii from hospitalized patients by both disk-diffusion and minimum inhibitory concentration (MIC) methods and to assess the rate of carbapenemase and MBL production among the isolates. MATERIALS AND METHODS: A. baumannii were identified from various clinical samples and antibiotic susceptibility profile was determined by the standard disk-diffusion method. Meropenem-resistant strains were tested further by agar dilution MIC for meropenem. Resistant isolates were screened for carbapenemase production by the modified Hodge test and positive isolates were further checked for metallo-ß-lacatmase production by the EDTA disk synergy test. RESULTS: 42 isolates (31.81%) showed resistance to meropenem by the disk diffusion method. 47.6% were carbapenemase positive by the modified Hodge test and 19% were MBL producers phenotypically by the EDTA disc synergy test (EDS). These meropenem-resistant isolates were resistant to most of the other antibiotics tested. These 42 isolates were recovered mostly from patients admitted to intensive care units. Four isolates of the A. baumannii complex were pan drug resistant and showed resistance to even tigecycline and polymyxin B. CONCLUSION: Carbapenem resistance has been increasingly reported, necessitating their detection. This study reports simple, carbapenemase, and MBL detection method that can be easily incorporated into the daily routine of a clinical laboratory.