A disk potentiation method using carbapenems as substrates and 3-aminophenyl boronic

A disk potentiation method using carbapenems as substrates and 3-aminophenyl boronic acid as an inhibitor was evaluated for the detection of carbapenemase (KPC)-type β-lactamases. of these organisms is difficult (5 20 Several methods have been developed specifically for the detection of KPC-producing ATCC 25922. The test strain is then streaked GSK1070916 radially through the GSK1070916 edge from the disk towards the periphery from the dish. After an over night incubation the current presence of a distorted inhibition area shows the carbapenem-hydrolyzing activity of the check strain. This technique is not too difficult to execute and feasible in medical laboratories but needs some encounter in interpreting the outcomes. A method calculating the hydrolysis of carbapenems by cell components continues to be reported aswell though this technique is technically demanding and GSK1070916 requires specialized laboratory equipment (15). Boronic acid compounds are known to be excellent inhibitors of class C β-lactamases (2 13 One such compound 3 boronic acid (APB) has recently been used successfully in detecting the production of plasmid-mediated class C β-lactamases in (6 10 12 23 We have subsequently undertaken a study investigating the effects of APB on zone diameters of carbapenem-containing disks in a set of isolates GSK1070916 producing KPC-type β-lactamases as well as other non-KPC broad-spectrum KRAS2 β-lactamases. A total of 23 epidemiologically unrelated and clinical isolates (10 producing KPC-type β-lactamase 3 ertapenem-resistant isolates without KPC-type β-lactamase 5 producing extended-spectrum β-lactamase [ESBLs] and 5 producing plasmid-mediated class C β-lactamase) were included in the study. Specifically the KPC-producing isolates originated from five hospitals in three states. We also included DH10B strains carrying recombinant plasmids that bear the genes for the metallo-β-lactamases (MBLs) IMP-1 and VIM-2. The β-lactamase types were determined by PCR analysis and nucleotide sequencing as appropriate. For the detection and sequencing of the KPC gene primers KPC-1-F (5′-GGC TTG CCG CTC GGT GAT ATT-3′) and KPC-1-R (5′-TAT CTG TGA GGG CGA AGG TTA-3′) were used at an annealing temperature of 62°C. ESBL genes and plasmid-mediated class C β-lactamase genes were amplified as described previously (11 18 The isolates were suspended in and diluted with normal saline to 108 CFU/ml by comparison with a McFarland 0.5 turbidity standard and spread on a Mueller-Hinton agar plate (BD Microbiology Systems Sparks MD) as recommended by the Clinical and Laboratory Standards Institute (CLSI) (8). The following disks (BD Microbiology Systems) were tested: ertapenem (10 μg) ertapenem (10 μg) with APB (300 μg) imipenem (10 μg) imipenem (10 μg) with APB (300 μg) meropenem (10 μg) meropenem (10 μg) with APB (300 μg) ceftazidime (30 μg) and ceftazidime (30 μg) with APB (300 μg). APB (3-aminophenyl boronic acid hydrochloride; Sigma-Aldrich St. Louis MO) was dissolved in water at 50 mg/ml and 6 μl was applied per disk. The amount of APB to be applied to the disks was determined based on the following observation: when the inhibitory effects of APB on representative KPC-positive and -negative isolates were examined at 100 200 300 450 and 600 μg per disk 300 μg was found to provide ideal level of sensitivity and specificity in discovering the current presence of KPC-type β-lactamase when coupled with ertapenem or meropenem and a cutoff of the 5-mm difference in area diameter was utilized. The area diameters had been read by at least two microbiologists. The customized Hodge check was performed to verify the creation of carbapenem-hydrolyzing β-lactamase as referred to previously (1). The full total email address details are summarized in Desk ?Desk1.1. The customized Hodge check was positive for many 10 KPC-producing medical isolates and 2 MBL-producing lab strains confirming the current presence of significant carbapenem-hydrolyzing activity. None of the other isolates had positive results with the modified Hodge test. All 10 KPC-producing isolates were resistant to ertapenem intermediate or resistant to meropenem and variably resistant to imipenem. Ertapenem and meropenem were both sensitive substrates for potentiation by APB. When APB was added to ertapenem or meropenem disks an increase in zone diameter of ≥5 mm was observed for all KPC-producing isolates (Table ?(Table1;1; Fig. ?Fig.1).1). Potentiation of ≥5 mm was observed in 6 of the 10 isolates for imipenem..