This phenotypic method is very helpful to detect carbapenemase production and provides a simple algorithm for the differentiation of KPC and MBL enzymes, especially in regions where KPC- and MBL-possessing Enterobacteriaceae are highly prevalent.

BSIs due to KPC-producing K. pneumoniae resulted in significantly increased mortality. The accurate and rapid detection of these pathogens is necessary for therapeutic considerations and for the implementation of infection control measures to contain them.

The worldwide increase in the occurrence and dissemination of KPC ␤-lactamases among gram-negative pathogens makes critical the early detection of these enzymes. Boronic acid disk tests using different antibiotic substrates were evaluated for detection of KPC-possessing Klebsiella pneumoniae isolates. A total of 57 genotypically confirmed KPC-possessing K. pneumoniae isolates with varying carbapenem MICs were examined. To measure the specificity of the tests, 106 non-KPC-possessing isolates (89 K. pneumoniae and 17 Escherichia coli isolates) were randomly selected among those exhibiting reduced susceptibility to cefoxitin, expanded-spectrum cephalosporins, or carbapenems. As many as 56, 53, and 40 of the non-KPC-possessing isolates harbored extended-spectrum ␤-lactamases, metallo-␤-lactamases, and plasmidmediated AmpC ␤-lactamases, respectively. By use of CLSI methodology and disks containing imipenem, meropenem, or cefepime, either alone or in combination with 400 g of boronic acid, all 57 KPC producers gave positive results (sensitivity, 100%) whereas all 106 non-KPC producers were negative (specificity, 100%). The meropenem duplicate disk with or without boronic acid demonstrated the largest differences in inhibition zone diameters between KPC producers and non-KPC producers. By use of disks containing ertapenem, all isolates were correctly differentiated except for five AmpC producers that gave falsepositive results (sensitivity, 100%; specificity, 95.3%). These practical and simple boronic acid disk tests promise to be very helpful for the accurate differentiation of KPC-possessing K. pneumoniae isolates, even in regions where different broad-spectrum ␤-lactamases are widespread.

The risk of malignancy in thyroid nodules increases in parallel with TSH concentrations within the normal range. TSH concentration at presentation is an independent predictor of thyroid malignancy.

A transferable ␤-lactamase produced by a multidrug-resistant clinical isolate of Enterobacter cloacae was studied. The bla gene was carried by a large (>80-kb) transmissible plasmid. Nucleotide sequence analysis of cloned fragments revealed that it was part of a gene cassette carried by a class 1 integron along with other resistance genes, including aac(6)-Ib. The encoded ␤-lactamase, designated IBC-1, was a novel class A enzyme that hydrolyzed ceftazidime and cefotaxime and was inhibited by tazobactam and, to a lesser extent, by clavulanate. Also, imipenem exhibited potent inhibitory activity against IBC-1. The enzyme consisted of 287 amino acid residues, including Ser-237, cysteines at positions 69 and 237a, and Arg-244, which may be implicated in its interaction with ␤-lactams. In amino acid sequence comparisons, IBC-1 displayed the highest similarity with the chromosomal penicillinase of Yersinia enterocolitica, a carbenicillinase from Proteus mirabilis GN79, the species-specific ␤-lactamases of Klebsiella oxytoca, and the carbapenemase Sme-1. However, a phylogenetic association with established ␤-lactamase clusters could not be conclusively shown.Production of extended-spectrum (ES) ␤-lactamases (ESBLs) is one of the major causes of resistance to the newer oxyimino-␤-lactams in enterobacteria. ␤-Lactamases hydrolyzing broadspectrum ␤-lactams and inhibited by clavulanic acid are considered to have ES properties (3). Except for the class D enzymes OXA-11 (9) and OXA-18 (22), which display ES properties, the ESBLs described to date belong to molecular class A. The most clinically important enzymes in this group are the ES descendants of the common plasmid-mediated TEM-1 and SHV-1 ␤-lactamases. Various non-TEM, non-SHV class A ␤-lactamases exhibiting ES activities have also been described. CTX-M-type (1, 8) and SFO-1 (13) ␤-lactamases can be placed in a cluster of clavulanate-inhibited enzymes that preferentially hydrolyze cefotaxime and are related to the species-specific ␤-lactamases of Klebsiella oxytoca (7) and other enterobacterial species. PER (2, 19) and VEB-1 (23) ␤-lactamases and their distant relatives CME-1 from Chryseobacterium meningosepticum (28) and the cephalosporinases of Bacteroides spp. (21) also exhibit ES properties. VEB-1 is the first class A ESBL that was found to be encoded by an integron-associated gene (23). Resistance genes found in the variable region of class 1 integrons are parts of cassettes (27) that also include an integrase-specific recombination segment called the 59-base element (59-be) (35). The bla genes that are most frequently associated with these structures belong to the pse, carb, and oxa types (27).In this work we describe IBC-1, a novel class A ESBL that was encoded by a class 1 integron-associated gene. The integron was located in a multidrug-resistant transferable plasmid found in an Enterobacter cloacae clinical strain. MATERIALS AND METHODSBacterial strains and plasmids. E. cloacae HT9 was isolated in January 1999 in Hippokration General Hospital, Thessaloniki, G...