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ABSTRACTThe current phenotypic methods for detecting carbapenemase-producing Enterobacteriaceae(CPE) allow differentiation between class A and B carbapenemases, but they cannot confirm in a single test class D OXA-48 carbapenemase producers. In this study, we evaluated a new phenotypic test, the OXA-48 disk test, which is based on an imipenem disk and two blank disks adjacent to the imipenem disk, loaded with the tested strain and impregnated with EDTA and EDTA plus phenyl boronic acid (PBA), respectively. The evaluation of the OXA-48 disk test was performed with 81 genotypically confirmed OXA-48-type-producing Enterobacteriaceaeisolates (41 extended-spectrum ß-lactamase [ESBL] producers, 3 AmpC producers, and 37 non-ESBL, non-AmpC producers). To measure the specificity of the test, 173 genotypically confirmed OXA-48-negative Enterobacteriaceaeisolates (57 Klebsiella pneumoniaecarbapenemase [KPC] producers, 34 VIM producers, 23 KPC/VIM producers, 22 NDM producers, and 37 AmpC or ESBL producers and porin deficient) that were nonsusceptible to at least one carbapenem were chosen for testing. Using the imipenem disk and the distortion of the inhibition halo around both blank disks containing EDTA and EDTA/PBA, the test differentiated all but 3 of the 81 OXA-48 producers (sensitivity of 96.3%). The test was negative for OXA-48 production in all but 4 of the 173 carbapenem-nonsusceptible isolates producing other carbapenemases, AmpCs, or ESBLs (specificity of 97.7%). This evaluation shows that the OXA-48 disk test is an accurate phenotypic method for the direct differentiation of OXA-48-producing Enterobacteriaceae. Its use along with combined disk tests employing inhibitor-supplemented carbapenem disks might allow the differentiation of the currently known carbapenemase types in Enterobacteriaceaespecies and provide important infection control information.

ABSTRACTThe worldwide dissemination of Enterobacteriaceaeproducing AmpC ß-lactamases and carbapenemases makes difficult the phenotypic detection of extended-spectrum ß-lactamases (ESBLs), as they may be masked by these additional enzymes. A modification of the CLSI ESBL confirmatory test was developed and evaluated in a comparative study for its ability to successfully detect ESBLs among Enterobacteriaceaeproducing various carbapenemases (Klebsiella pneumoniaecarbapenemase [KPC], VIM, NDM, and OXA-48) and plasmidic or derepressed AmpCs. The modified CLSI ESBL confirmatory test was performed with cefotaxime and ceftazidime disks with and without clavulanate, on which both boronic acid (BA) and EDTA were dispensed. A total of 162 genotypically confirmed ESBL-positive Enterobacteriaceaeisolates (83 carbapenemase/ESBL producers, 25 AmpC/ESBL producers, and 54 ESBL-only producers) were examined. For comparison, 139 genotypically confirmed ESBL-negative Enterobacteriaceaeisolates (94 of them possessed carbapenemases and 20 possessed AmpCs) were also tested. The standard CLSI ESBL confirmatory test was positive for 106 of the 162 ESBL producers (sensitivity, 65.4%) and showed false-positive results for 4 of the 139 non-ESBL producers (specificity, 97.1%). The modified CLSI ESBL confirmatory test detected 158 of 162 ESBL producers (sensitivity, 97.5%) and showed no false-positive results for non-ESBL producers (specificity, 100%). The findings of the study demonstrate that the modified CLSI ESBL confirmatory test using antibiotic disks containing both BA and EDTA accurately detects ESBLs in Enterobacteriaceaeregardless of the coexistence of additional ß-lactam resistance mechanisms.

ABSTRACTTwo colistin-susceptible/colistin-resistant (Cols/Colr) pairs of Acinetobacter baumanniistrains assigned to international clone 2, which is prevalent worldwide, were sequentially recovered from two patients after prolonged colistin administration. Compared with the respective Colsisolates (Ab248 and Ab299, both having a colistin MIC of 0.5 μg/ml), both Colrisolates (Ab249 and Ab347, with colistin MICs of 128 and 32 μg/ml, respectively) significantly overexpressed pmrCABgenes, had single-amino-acid shifts in the PmrB protein, and exhibited significantly slower growth. The Colrisolate Ab347, tested by proteomic analysis in comparison with its Colscounterpart Ab299, underexpressed the proteins CsuA/B and C from the csuoperon (which is necessary for biofilm formation). This isolate also underexpressed aconitase B and different enzymes involved in the oxidative stress response (KatE catalase, superoxide dismutase, and alkyl hydroperoxide reductase), suggesting a reduced response to reactive oxygen species (ROS) and, consequently, impaired colistin-mediated cell death through hydroxyl radical production. Colsisolates that were indistinguishable by macrorestriction analysis from Ab299 caused six sequential bloodstream infections, and isolates indistinguishable from Ab248 caused severe soft tissue infection, while Colrisolates indistinguishable from Ab347 and Ab249 were mainly colonizers. In particular, a Colsisolate identical to Ab299 was still invading the bloodstream 90 days after the colonization of this patient by Colrisolates. These observations indicate considerably lower invasiveness of A. baumanniiclinical isolates following the development of colistin resistance.

ABSTRACTAlthough numerous studies have documented outbreaks of carbapenem-resistant Klebsiella pneumoniae(CRKP) possessing various carbapenemases, reports on outbreaks due to CRKP possessing extended-spectrum ß-lactamases (ESBLs) and/or AmpCs with porin lesions have been limited. Here, we describe an outbreak caused by an ertapenem-resistant, CTX-M-15-producing clonal K. pneumoniaestrain expressing an OmpK36 porin variant. From May 2012 to November 2012, 37 ertapenem-resistant K. pneumoniaeisolates phenotypically negative for carbapenemase production were recovered from 19 patients hospitalized in the intensive care unit of a Greek hospital. The isolates were either susceptible or intermediate to other carbapenems and resistant to all remaining ß-lactams but cefotetan. Phenotypic and molecular analysis revealed the presence in all isolates of the blaCTX-M-15gene on a conjugative 100-kb plasmid, disruption in the expression of the ompK35gene, and the production of an Ompk36 porin variant. The index case was a patient admitted from another hospital. Active surveillance upon admission and on a weekly basis was immediately initiated; environmental samples were also periodically tested. Molecular typing showed that all clinical isolates as well as two ertapenem-resistant environmental K. pneumoniaeisolates belonged to the same clonal type and were assigned to multilocus sequence typing (MLST) sequence type 101 (ST101). As all colonized/infected patients were hospitalized during overlapping periods, cross-infection was considered the main route for the dissemination of the outbreak strain. Despite reinforcement of infection control measures and active surveillance, the outbreak lasted approximately 7 months. Identification of hidden carriers upon admission and by screening on a weekly basis was found valuable for early recognition and subsequent successful management of the outbreak.

ABSTRACTCarbapenemase-producing Enterobacteriaceae(CPE) are rapidly spreading worldwide. Early detection of fecal CPE carriers is essential for effective infection control. Here, we evaluated the performance of a meropenem combined disk test (CDT) for rapidly differentiating CPE isolates directly from rectal swabs. The screening method was applied for 189 rectal swabs from hospitalized patients at high risk for CPE carriage. Swabs were suspended in 1 ml saline and cultured for confluent growth onto a MacConkey agar plate with a meropenem (MER) disk alone, a MER disk plus phenyl boronic acid (PBA), a MER disk plus EDTA, and a MER disk plus PBA and EDTA. An inhibition zone of =25 mm around the MER disk alone indicated carriage of carbapenem-resistant organisms. Furthermore, =5-mm differences in the inhibition zone between MER disks without and with the inhibitors (PBA, EDTA, or both) were considered positive results for detecting Klebsiella pneumoniaecarbapenemase (KPC), metallo-ß-lactamase (MBL), or both carbapenemases, respectively. For comparison, rectal suspensions were tested using MacConkey plates with ertapenem (MacERT) disks and PCR (PCR-S) for carbapenemase genes. Of the 189 samples, 97 were genotypically confirmed as CPE positive by one of the three protocols tested. The CDT, MacERT disks, and PCR-S assays exhibited sensitivities of 94.8%, 96.9%, and 94.8% and specificities of 100%, 98.9%, and 100%, respectively, for detecting CPE-positive swabs. Moreover, the CDT correctly differentiated the production of KPC, MBL, or both carbapenemases in 78 of the 97 (80.4%) CPE-positive rectal swabs. Our results demonstrate that the CDT may provide a simple and inexpensive method for detecting and differentiating the carbapenemase type within a single day without requiring further testing and additional delay, supporting the timely implementation of infection control measures.

ABSTRACTThe recent emergence of carbapenemase-producing Enterobacteriaceaestrains represents a major threat for hospitalized patients. We document the dissemination and control of carbapenemase-producing Klebsiella pneumoniaeclones in a Greek hospital. During a 3-year study period (January 2009 to December 2011), carbapenemase-producing K. pneumoniaestrains were isolated from clinical samples from 73 individual patients. Phenotyping and molecular testing confirmed that 52 patients were infected with K. pneumoniaecarbapenemase 2 (KPC-2) producers, 12 were infected with VIM-1 producers, and the remaining 9 were infected with isolates producing both KPC-2 and VIM-1 enzymes. Twenty-eight of these clinical cases were characterized as imported health care associated, and 23 of these were attributed to KPC producers and 5 were attributed to KPC and VIM producers. The remaining 45 cases were deemed hospital acquired. In the second year of the study, intensified infection control intervention was implemented, followed by active surveillance and carrier isolation in the third year. The incidence of carbapenemase-producing K. pneumoniaepatient cases decreased from 0.52/1,000 patient days in 2009 to 0.32/1,000 patient days in 2010 (P= 0.075). Following these additional infection control measures, the incidence fell to 0.21/1,000 patient days in 2011 and differed significantly from that in 2009 (P= 0.0028). Despite the fact that the imported cases of carbapenemase-producing K. pneumoniaewere equally distributed over this 3-year period, the incidence of hospital-acquired cases decreased from 0.36/1,000 patient days in 2009 to 0.19/1,000 patient days in 2010 (P= 0.058) and to 0.1/1,000 patient days in 2011 (P= 0.0012). Our findings suggest that rigorous infection control measures and active surveillance can effectively reduce the incidence of secondary transmission due to KPC-producing pathogens.

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.

ABSTRACTThe aim of the present study was to investigate the epidemiological link of multidrug-resistant Klebsiella oxytocaisolates causing community-onset infections among patients attending our outpatient department and to investigate the underlying resistance mechanisms. The isolates were tested by agar dilution MICs, phenotypic carbapenemase testing, enterobacterial repetitive intergenic consensus-PCR, and pulsed-field gel electrophoresis (PFGE). PCR assays and nucleotide sequencing were employed for the identification of blagene types and the mapping of the integron-containing metallo-β-lactamase (MBL) gene. During the study period (January 2005 to April 2007), nine broad-spectrum cephalosporin-resistant K. oxytocaclinical isolates were prospectively collected from separate outpatients with urinary tract infections. In all cases, the patients had been hospitalized or exposed to health care facilities during the preceding year. Molecular typing revealed that all isolates belonged to the same K. oxytocaclonal type, which contained five PFGE subtypes. A novel chromosomal OXY-2 β-lactamase type variant (OXY-2-9) was detected in all isolates, but no mutations in the promoter region justifying blaOXYgene overproduction were detected. In addition, all isolates harbored the plasmidic CMY-31 (LAT-4) AmpC cephalosporinase, while three of them harbored VIM-1 MBL in a class 1 integron structure. This is the first study to present the dissemination in the community of multidrug-resistant K. oxytocaisolates causing extrahospital infections.

ABSTRACTMeropenem heteroresistance was investigated in six apparently meropenem-susceptible, Klebsiella pneumoniaecarbapenemase (KPC)-producing K. pneumoniae(KPC-KP) clinical isolates, compared with that in carbapenemase-negative, meropenem-susceptible controls. In population analyses, the KPC-KP isolates grew at meropenem concentrations of 64 to 256 µg/ml. Heteroresistant colonies had significantly elevated expression of the blaKPCgene compared with the native populations but did not retain heteroresistance when subcultured in drug-free media. Time-kill assays indicated that meropenem alone was not bactericidal against KPC-KP but efficiently killed the control strains.

ABSTRACTThree patients admitted to a Greek hospital were infected with Serratia marcescensisolates that exhibited reduced susceptibility to carbapenems and harbored Klebsiella pneumoniaecarbapenemase (KPC) enzymes. In two of these cases, the patients were initially infected by carbapenem-susceptible S. marcescensisolates. Molecular typing and plasmid analysis suggested that all three patients had clonally indistinguishable isolates of S. marcescensthat acquired a plasmid-mediated blaKPC-2gene during the hospitalization.

Three patients admitted to a Greek hospital were infected with Serratia marcescens isolates that exhibited reduced susceptibility to carbapenems and harbored Klebsiella pneumoniae carbapenemase (KPC) enzymes. In two of these cases, the patients were initially infected by carbapenem-susceptible S. marcescens isolates. Molecular typing and plasmid analysis suggested that all three patients had clonally indistinguishable isolates of S. marcescens that acquired a plasmid-mediated blaKPC-2gene during the hospitalization.

ABSTRACTOXA-48-like carbapenemases have only recently emerged in Europe. OXA-162 is a rare OXA-48 variant usually coexpressed with extended-spectrum β-lactamases. Here, we report the identification of the first OXA-162 carbapenemase-producing Klebsiella pneumoniaeisolates, which coexpressed an AmpC cephalosporinase (DHA-1), retrieved from a patient in Greece. They belonged to a single sequence type (ST11) and caused the first documented community-onset urinary tract infections attributable to an OXA-48-like-producing Enterobacteriaceaestrain.