Ioulia Kristo

Evaluation of Boronic Acid Disk Tests for Differentiating KPC-Possessing Klebsiella pneumoniae Isolates in the Clinical Laboratory

ABSTRACT The worldwide increase in the occurrence and dissemination of KPC β-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 β-lactamases, metallo-β-lactamases, and plasmid-mediated AmpC β-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. pneumoniae isolates, even in regions where different broad-spectrum β-lactamases are widespread.

Clonal spread of KPC-2 carbapenemase-producing Klebsiella pneumoniae strains in Greece

OBJECTIVES KPC-possessing Klebsiella pneumoniae have been found to be widespread in several regions but are still rarely detected in Europe. We describe the characteristics of an outbreak caused by KPC producers in a tertiary care Greek hospital. METHODS During a 12 month period (October 2007-September 2008), 47 patients in Hippokration University Hospital yielded K. pneumoniae isolates that exhibited reduced susceptibility to carbapenems and were phenotypically positive for carbapenemase production but negative for metallo-beta-lactamase (MBL) production. Single patient isolates were tested by Vitek 2, Etest, agar dilution MICs, phenotypic assays and PFGE. Carbapenemase and other beta-lactamase genes were identified by PCR and sequencing. Patient records were retrospectively reviewed to access co-morbidities, antibiotic exposure prior to infection and outcome. RESULTS The 47 K. pneumoniae isolates exhibited various susceptibilities to imipenem and meropenem; all were non-susceptible to ertapenem and several other antibiotics but most were susceptible to gentamicin, colistin and tigecycline. PFGE classified the isolates into two clonal types, with the predominant type, which was closely related to that of hyperepidemic strains from the USA and Israel, comprising three subtypes. All isolates carried the bla(KPC-2) gene; 45 also carried bla(SHV-12) and 29 bla(TEM-1). Patients were hospitalized in nine different units. The median length of hospital stay prior to KPC isolation was 21 days; 38 patients (80.9%) had evidence of clinical infection due to a KPC producer and 16 (34%) had bacteraemia. The crude mortality rate was 27.7%. A beta-lactam/beta-lactamase inhibitor combination was the most frequently administered antimicrobial prior to KPC isolation (20 patients; 42.5%), whereas only nine patients (19.1%) had prior carbapenem use. CONCLUSIONS This study presents for the first time a wide intrahospital spread of KPC-producing K. pneumoniae clones in a European hospital. The KPC producers were rapidly disseminated in several units, indicating the difficulty in restraining such multidrug-resistant clones when they have been established in a hospital environment.

Outbreaks in Distinct Regions Due to a Single Klebsiella pneumoniae Clone Carrying a blaVIM-1 Metallo-β-Lactamase Gene

ABSTRACT From December 2004 to March 2005, 27 Klebsiella pneumoniae clinical 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-1 metallo-β-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. pneumoniae strains in distinct regions where up to now blaVIM-2 and blaVIM-4 alleles were common is worrisome.

First occurrence of KPC-2-possessing Klebsiella pneumoniae in a Greek hospital and recommendation for detection with boronic acid disc tests

OBJECTIVES To investigate the first KPC carbapenemase-producing Klebsiella pneumoniae isolate from a Greek hospital, including phenotypic methods to aid recognition of this resistance type. METHODS A carbapenem-resistant clinical isolate of K. pneumoniae was recovered from a hospitalized Greek patient. Detailed susceptibility testing was carried out by the agar dilution method. The isolate was screened by phenotypic and genotypic assays for the presence of various beta-lactamases. Boronic acid disc tests were performed to show the ability of these tests to detect production of the KPC enzymes. The potential for conjugal transfer of carbapenem resistance was examined by biparental matings, plasmid analysis and PCR studies. RESULTS The isolate possessed on the same self-transferable plasmid the KPC-2 carbapenemase and the SHV-12 extended-spectrum beta-lactamase. Although the isolate did not produce an AmpC-type enzyme, the production of KPC-2 was associated with positive boronic acid disc tests using cephamycins and cefotaxime as well as cefepime and carbapenems as substrates. DISCUSSION KPC-2-possessing K. pneumoniae clinical isolates seem to have been introduced in our region. Boronic acid disc tests using boronic acid in combination with carbapenems or cefepime may help the phenotypic detection of KPC enzymes and their distinction from plasmid-mediated AmpC enzymes.

Detection of the new metallo-β-lactamase VIM-19 along with KPC-2, CMY-2 and CTX-M-15 in Klebsiella pneumoniae

OBJECTIVES To report the identification of the metallo-beta-lactamase (MBL) variant VIM-19 in a Klebsiella pneumoniae clinical strain co-producing KPC-2 carbapenemase, CMY-2 cephalosporinase and CTX-M-15 extended-spectrum beta-lactamase. METHODS MICs were determined by agar dilution. Phenotypic tests were performed to detect carbapenemase production. PCR and nucleotide sequencing were used for the identification of bla gene types and mapping of the integron carrying the MBL gene. The location of the MBL and KPC alleles was investigated by mating experiments, plasmid analysis and PCR assays. RESULTS Imipenem, meropenem and ertapenem MICs for the study strain were 32, 16 and 64 mg/L, respectively. The strain carried bla(TEM-1), bla(CMY-2), bla(KPC-2) and bla(CTX-M-15) genes along with the gene bla(VIM-19), which was located in a class 1 integron as the first gene cassette, followed by aacA6, dfrA1 and aadA1 cassettes. Mating experiments, plasmid analysis and PCR assays revealed that bla(VIM-19) and bla(CMY-2) were carried on an approximately 150 kb self-transferable plasmid, while bla(KPC-2) and bla(TEM-1) were on an approximately 70 kb self-transferable plasmid; bla(CTX-M-15) was non-transferable. CONCLUSIONS The detection of the new MBL, VIM-19, which has enhanced carbapenemase activity, along with KPC-2, CMY-2 and CTX-M-15 is of concern. Further spread of the respective strains or plasmids may have serious consequences for antimicrobial chemotherapy.

Growth Retardation, Reduced Invasiveness, and Impaired Colistin-Mediated Cell Death Associated with Colistin Resistance Development in Acinetobacter baumannii

ABSTRACT Two colistin-susceptible/colistin-resistant (Cols/Colr) pairs of Acinetobacter baumannii strains assigned to international clone 2, which is prevalent worldwide, were sequentially recovered from two patients after prolonged colistin administration. Compared with the respective Cols isolates (Ab248 and Ab299, both having a colistin MIC of 0.5 μg/ml), both Colr isolates (Ab249 and Ab347, with colistin MICs of 128 and 32 μg/ml, respectively) significantly overexpressed pmrCAB genes, had single-amino-acid shifts in the PmrB protein, and exhibited significantly slower growth. The Colr isolate Ab347, tested by proteomic analysis in comparison with its Cols counterpart Ab299, underexpressed the proteins CsuA/B and C from the csu operon (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. Cols isolates that were indistinguishable by macrorestriction analysis from Ab299 caused six sequential bloodstream infections, and isolates indistinguishable from Ab248 caused severe soft tissue infection, while Colr isolates indistinguishable from Ab347 and Ab249 were mainly colonizers. In particular, a Cols isolate identical to Ab299 was still invading the bloodstream 90 days after the colonization of this patient by Colr isolates. These observations indicate considerably lower invasiveness of A. baumannii clinical isolates following the development of colistin resistance.

Characteristics of Meropenem Heteroresistance in Klebsiella pneumoniae Carbapenemase (KPC)-Producing Clinical Isolates of K. pneumoniae

ABSTRACT Meropenem heteroresistance was investigated in six apparently meropenem-susceptible, Klebsiella pneumoniae carbapenemase (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 bla KPC gene 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.

Large Dissemination of VIM-2-Metallo-β-Lactamase-Producing Pseudomonas aeruginosa Strains Causing Health Care-Associated Community-Onset Infections

ABSTRACT During a 3-year period (May 2005 to April 2008), a series of 45 outpatients presented with community-onset urinary tract infections due to carbapenem-resistant Pseudomonas aeruginosa isolates. Forty of them had a history of previous hospitalization or exposure to healthcare facilities, while the remaining five had not been previously admitted to our healthcare facilities or elsewhere within the preceding 12 months. In 18 outpatients, the carbapenem-resistant organisms caused recurrent community-onset urinary tract infections, while in three outpatients the organisms were also implicated in bacteremic episodes. All 45 single-patient P. aeruginosa isolates harbored the blaVIM-2 metallo-β-lactamase (MBL) gene in a common class 1 integron structure. They belonged to one predominant pulsed-field gel electrophoresis type and three sporadically detected types; two of the sporadic clonal types were identified among outpatients without previous exposure to healthcare facilities, while the predominant clonal type was also identified to cause infections in hospitalized patients. This is the first study documenting that MBL-producing P. aeruginosa isolates cause community-onset infections that are related or not with exposure to healthcare facilities. Community-onset infections in our patients most likely resulted from the nosocomial acquisition of MBL producers, followed by a prolonged digestive carriage. The high rate of recurrent infections in the community underlies the difficulty of constraining infections caused by such microorganisms in the extrahospital setting.

Intensive care unit dissemination of multiple clones of linezolid-resistant Enterococcus faecalis and Enterococcus faecium

OBJECTIVES Outbreaks caused by linezolid-resistant (LR) enterococci remain rare. We report the epidemiological and molecular characteristics of the multiclonal dissemination of LR enterococci in the intensive care unit (ICU) of a Greek hospital. METHODS All LR enterococcal isolates recovered from patients hospitalized in the ICU of the University Hospital of Larissa, Greece, between January 2007 and October 2008 were included. Isolates were tested by PFGE and PCR followed by sequence analysis of the entire 23S rRNA gene. Patient records were retrieved to access patterns of acquisition and outcome. RESULTS Sixteen separate patients were infected and/or colonized by 22 LR enterococcal isolates (17 Enterococcus faecium and 5 Enterococcus faecalis). Linezolid MICs varied from 8 to 16 mg/L; 12 isolates showed cross-resistance to vancomycin. Genotyping revealed as many as seven and three PFGE types among E. faecium and E. faecalis isolates, respectively, indicating multiclonal spread of LR enterococci. Nine patients had received linezolid prior to the recovery of LR enterococci, while the remaining seven patients were not exposed to the drug. All isolates carried the mutation G2576T; the mutated position was heterogeneous in 12 isolates and homogeneous in 10. CONCLUSIONS The multiclonal composition of LR enterococci indicates that linezolid resistance possibly occurred on several independent occasions. Its acquisition was often not related to linezolid administration; patients might have acquired their LR isolate from another patient that had received linezolid or, alternatively, resistance may have arisen by mutation that occurred independently.

A Combined Disk Test for Direct Differentiation of Carbapenemase-Producing Enterobacteriaceae in Surveillance Rectal Swabs

ABSTRACT Carbapenemase-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 pneumoniae carbapenemase (KPC), metallo-β-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.