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ABSTRACTThe accurate phenotypic detection of Klebsiella pneumoniaecarbapenemase (KPC)-producing Enterobacteriaceaeis an increasing necessity worldwide. We evaluated the performance of boronic acid combined-disk tests using as substrate imipenem or meropenem and as inhibitor of KPC production 300 µg aminophenylboronic acid (APBA), 600 µg APBA, or 400 µg phenylboronic acid (PBA). Tests were considered positive when an increase in the growth-inhibitory zone around a carbapenem disk with KPC inhibitor was 5 mm or greater of the growth-inhibitory zone diameter around the disk containing carbapenem alone. The comparison of the combined-disk tests was performed with 112 genotypically confirmed KPC-possessing Enterobacteriaceaeisolates. To measure the specificity of the tests, 127 genotypically confirmed KPC-negative Enterobacteriaceaeisolates that were nonsusceptible to at least one carbapenem were chosen for testing. Using disks containing imipenem without and with 300 µg APBA, 600 µg APBA, or 400 µg PBA, 72, 92, and 112 of the KPC producers, respectively, gave positive results (sensitivities, 64.3%, 82.1%, and 100%, respectively). Using disks containing meropenem without and with 300 µg APBA, 600 µg APBA, or 400 µg PBA, 87, 108, and 112 of the KPC producers, respectively, gave positive results (sensitivities, 77.7%, 96.4%, and 100%, respectively). Among KPC producers, the disk potentiation tests using meropenem and PBA demonstrated the largest differences in inhibition zones (P< 0.001). All combined-disk tests correctly identified 124 of the 127 non-KPC producers (specificity, 97.6%). This comparative study showed that PBA is the most effective inhibitor of KPC enzymes, and its use in combined-disk tests with meropenem may give the most easily interpreted results.

ABSTRACTWe evaluated boronic acid (BA)-based methods for their ability to detect extended-spectrum ß-lactamases (ESBLs) among clinical isolates of KPC-producing members of the Enterobacteriaceaefamily. A total of 155 isolates of Klebsiella pneumoniae(n= 141), Escherichia coli(n= 6), Enterobacter aerogenes(n= 6), and Klebsiella oxytoca(n= 2) genotypically confirmed to be KPC producers were analyzed. As many as 118 isolates harbored ESBLs (103 harbored SHV-type ESBLs, 13 harbored CTX-M-type ESBLs, and 2 harbored both SHV- and CTX-M-type ESBLs); the remaining 37 isolates were genotypically negative for ESBL production. The CLSI ESBL confirmatory test was positive for 79 of the 118 ESBL producers (sensitivity, 66.9%), while all 37 non-ESBL producers were negative (specificity, 100%). When a =5-mm increase in the zone diameter of either the cefotaxime (CTX)-clavulanate (CA) or the ceftazidime (CAZ)-CA disks containing BA compared with the zone diameter of the CTX or CAZ disks containing BA was considered to be a positive result for ESBL production, the method detected all 118 ESBL producers (sensitivity, 100%) and showed no false-positive results for non-ESBL producers (specificity, 100%). Double-disk synergy tests, in which disks of CTX, CAZ, aztreonam, or cefepime in combination with BA were placed at distances of 20, 25, and 30 mm (center to center) from a disk containing amoxicillin (amoxicilline)-clavulanate-BA, were able to detect 116 (98.3%), 101 (85.6%), and 28 (23.7%) of the ESBL-positive isolates, respectively; no false-positive results for non-ESBL-producing isolates were detected. Our results demonstrate that the modified CLSI ESBL confirmatory test with antibiotic disks containing BA is the most accurate phenotypic method for the detection of ESBLs in Enterobacteriaceaeproducing KPC carbapenemases.

ABSTRACTThe worldwide increase in the occurrence and dissemination of KPC ß-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 pneumoniaeisolates. A total of 57 genotypically confirmed KPC-possessing K. pneumoniaeisolates with varying carbapenem MICs were examined. To measure the specificity of the tests, 106 non-KPC-possessing isolates (89 K. pneumoniaeand 17 Escherichia coliisolates) 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 ß-lactamases, metallo-ß-lactamases, and plasmid-mediated AmpC ß-lactamases, respectively. By use of CLSI methodology and disks containing imipenem, meropenem, or cefepime, either alone or in combination with 400 µg 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 false-positive 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. pneumoniaeisolates, even in regions where different broad-spectrum ß-lactamases are widespread.

ABSTRACTFrom November 2006 to April 2007, nine nonrepetitive isolates of Klebsiella pneumoniaewith reduced susceptibility or resistance to carbapenems were recovered from clinical specimens from separate patients hospitalized in a tertiary care hospital. The imipenem-EDTA synergy test was positive for all isolates. PCR, sequencing, and transferability experiments revealed the novel blaVIM-12metallo-ß-lactamase gene, which was plasmid mediated and located in a class 1 integron. Pulsed-field gel electrophoresis demonstrated a single macrorestriction pattern, indicating the clonal spread of VIM-12-producing K. pneumoniae.

ABSTRACTFrom December 2004 to March 2005, 27 Klebsiella pneumoniaeclinical isolates that were positive by the imipenem-EDTA double-disk synergy test and that exhibited a single macrorestriction pattern were recovered in two distinct Greek hospitals. The isolates carried a transferable blaVIM-1metallo-ß-lactamase gene in a class 1 integron. Reverse transcriptase PCR showed that the gene was similarly expressed in low- and high-level carbapenem-resistant isolates, indicating the existence of additional resistance mechanisms. The clonal spread of VIM-1-producing K. pneumoniaestrains in distinct regions where up to now blaVIM-2and blaVIM-4alleles were common is worrisome.

ABSTRACTFrom June to July 1998, two episodes of Candida tropicalisfungemia occurred in the Aristotle University neonatal intensive care unit (ICU). To investigate this uncommon event, a prospective study of fungal colonization and infection was conducted. From December 1998 to December 1999, surveillance cultures of the oral cavities and perinea of the 593 of the 781 neonates admitted to the neonatal ICU who were expected to stay for >7 days were performed. Potential environmental reservoirs and possible risk factors for acquisition of C. tropicaliswere searched for. Molecular epidemiologic studies by two methods of restriction fragment length polymorphism analysis and two methods of random amplified polymorphic DNA analysis were performed. Seventy-two neonates were colonized by yeasts (12.1%), of which 30 were colonized by Candida albicans, 17 were colonized by C. tropicalis, and 5 were colonized by Candida parapsilosis. From December 1998 to December 1999, 10 cases of fungemia occurred; 6 were due to C. parapsilosis, 2 were due to C. tropicalis, 1 was due to Candida glabrata, and 1 was due to Trichosporon asahii(12.8/1,000 admissions). Fungemia occurred more frequently in colonized than in noncolonized neonates (P< 0.0001). Genetic analysis of 11 colonization isolates and the two late blood isolates of C. tropicalisdemonstrated two genotypes. One blood isolate and nine colonization isolates belonged to a single type. The fungemia/colonization ratio of C. parapsilosis(3/5) was greater than that of C. tropicalis(2/17, P= 0.05), other non-C. albicans Candidaspp. (1/11, P= 0.02), or C. albicans (0/27, P= 0.05). Extensive environmental cultures revealed no common source of C. tropicalisor C. parapsilosis. There was neither prophylactic use of azoles nor other risk factors found for acquisition of C. tropicalisexcept for total parenteral nutrition. A substantial risk of colonization by non-C. albicans Candidaspp. in the neonatal ICU may lead to a preponderance of C. tropicalisas a significant cause of neonatal fungemia.

ABSTRACTThe aim of the present study was to investigate the frequency of extended-spectrum ß-lactamases (ESBLs) in a consecutive collection of clinical isolates of Enterobacterspp. The abilities of various screening methods to detect ESBLs in enterobacters were simultaneously tested. Among the 68 consecutive isolates (56Enterobacter cloacaeand 12 Enterobacter aerogenesisolates) that were analyzed for ß-lactamase content, 21 (25 and 58%, respectively) possessed transferable ESBLs with pIs of 8.2 and phenotypic characteristics of SHV-type enzymes, 8 (14.3%) of the E. cloacaeisolates produced a previously nondescribed, clavulanate-susceptible ESBL that exhibited a pI of 6.9 and that conferred a ceftazidime resistance phenotype on Escherichia colitransconjugants, and 2 E. cloacaeisolates produced both of these enzymes. Among the total of 31 isolates that were considered ESBL producers, the Vitek ESBL detection test was positive for 2 (6.5%) strains, and the conventional double-disk synergy test (DDST) with amoxicillin-clavulanate and with expanded-spectrum cephalosporins and aztreonam was positive for 5 (16%) strains. Modifications of the DDST consisting of closer application of the disks (at 20 instead of 30 mm), the use of cefepime, and the use of both modifications increased the sensitivity of this test to 71, 61, and 90%, respectively. Of the 37 isolates for which isoelectric focusing failed to determine ESBLs, the Vitek test was false positive for 1 isolate and the various forms of DDSTs were false-positive for 3 isolates.

ABSTRACTBetween January and December 1999, in Hippokration General Hospital, Thessaloniki, Greece, a large proportion of the methicillin-resistant Staphylococcus aureusisolates (34.4%) exhibited susceptibility to virtually all alternative non-ß-lactam antibiotics, including tobramycin. Twenty-five of them were selected randomly for further testing; all belonged to a unique genotype and were characterized as heterogeneously resistant to oxacillin. The aadDgene, encoding tobramycin resistance, failed to be amplified in all cases, indicating absence of the gene or the entire plasmid pUB110 from the mecDNA.

ABSTRACTIntroduction of the Vitek GNS-506 susceptibility testing cards in the Hippokration General Hospital, Thessaloniki, Greece, resulted in an apparently high prevalence of imipenem-resistant Acinetobacter baumannii. When 35 of these isolates were further tested by disk diffusion, broth microdilution, and agar dilution assays, 32 were imipenem sensitive by all tests and three were sensitive or intermediate, depending on the method. The pseudoresistant acinetobacters did not form a genetically homogeneous group. It is suggested that the detection of imipenem-resistant A. baumanniiisolates by this system should be confirmed by an additional susceptibility test.

ABSTRACTRoseomonasis a newly described genus of pink-pigmented, nonfermentative, gram-negative bacteria that have been recognized as a cause of human infections. Roseomonas fauriaeis a species rarely isolated from clinical specimens. We report the first known case of peritonitis caused by R. fauriaein a patient receiving continuous ambulatory peritoneal dialysis.

ABSTRACTReports of serious infections caused by Ewingella americanahave been rare. A case of E. americanaperitonitis in a patient receiving continuous ambulatory peritoneal dialysis is described. This is the first report of E. americanacausing such an infection.