Olympia Zarkotou

Risk Factors and Outcomes Associated with Acquisition of Colistin-Resistant KPC-Producing Klebsiella pneumoniae: a Matched Case-Control Study

ABSTRACT A matched 1:3 case-control study investigated factors predicting colistin-resistant versus colistin-susceptible KPC-producing Klebsiella pneumoniae acquisition and its impact on patient outcomes. Case patients were more often admitted from other institutions (P = 0.019) and had longer therapy with β-lactam/β-lactamase inhibitors (P = 0.002) and higher overall mortality (P = 0.05). All 52 study isolates were clonally related, suggesting horizontal dissemination. None of these parameters independently predicted colistin resistance, which probably occurred in a susceptible KPC-KP strain that was subsequently disseminated horizontally.

Outbreak of OXA-48 carbapenemase-producing Klebsiella pneumoniae in Greece involving an ST11 clone

OBJECTIVES First detected in Enterobacteriaceae isolates in Turkey, the OXA-48 carbapenemase has gradually disseminated in the wider Mediterranean area and Europe. Despite reports from other European regions, until now no such isolates have been detected in Greece. We describe the characteristics of the first outbreak caused by OXA-48-producing Klebsiella pneumoniae in Greece. METHODS From December 2011 to March 2012, 13 ertapenem-resistant K. pneumoniae isolates, which were positive by the modified Hodge test while remaining negative by phenotypic screening for metallo-β-lactamase (MBL) and KPC production, were recovered from nine patients. Patient records were retrieved to access patterns of acquisition. Resistance genes were identified by PCR and sequencing. ompK35, ompK36 and the genetic environment of the bla(OXA-48) gene were investigated. Plasmid profiling, conjugation experiments, PFGE and multilocus sequence typing (MLST) were performed. RESULTS All isolates harboured the bla(OXA-48) gene along with the bla(CTX-M-15) and bla(OXA-1) genes. The bla(OXA-48) gene was located on a self-transferable IncL/M-type plasmid of ~62 kb, which harboured no other resistance genes. IS1999 was located upstream of the bla(OXA-48) gene. Genetic disruptions of the ompK35 and ompK36 genes were not detected. The isolates belonged to a unique PFGE clone and MLST assigned them to sequence type ST11. All cases were characterized as hospital acquired and none of them was linked to immigration or history of travel in endemic areas. CONCLUSIONS Carbapenem resistance due to MBL and KPC carbapenemases is currently on an endemic scale in Greece and this report highlights the wider undetected dissemination of yet another carbapenemase in this region.

Comparative Evaluation of Colistin Susceptibility Testing Methods among Carbapenem-Nonsusceptible Klebsiella pneumoniae and Acinetobacter baumannii Clinical Isolates

ABSTRACT We compared six colistin susceptibility testing (ST) methods on 61 carbapenem-nonsusceptible Klebsiella pneumoniae (n = 41) and Acinetobacter baumannii (n = 20) clinical isolates with provisionally elevated colistin MICs by routine ST. Colistin MICs were determined by broth microdilution (BMD), BMD with 0.002% polysorbate 80 (P80) (BMD-P80), agar dilution (AD), Etest, Vitek2, and MIC test strip (MTS). BMD was used as the reference method for comparison. The EUCAST-recommended susceptible and resistant breakpoints of ≤2 and >2 μg/ml, respectively, were applied for both K. pneumoniae and A. baumannii. The proportions of colistin-resistant strains were 95.1, 77, 96.7, 57.4, 65.6, and 98.4% by BMD, BMD-P80, AD, Etest, MTS, and Vitek2, respectively. The Etest and MTS methods produced excessive rates of very major errors (VMEs) (39.3 and 31.1%, respectively), while BMD-P80 produced 18% VMEs, AD produced 3.3% VMEs, and Vitek2 produced no VMEs. Major errors (MEs) were rather limited by all tested methods. These data show that gradient diffusion methods may lead to inappropriate colistin therapy. Clinical laboratories should consider the use of automated systems, such as Vitek2, or dilution methods for colistin ST.

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.

Comparative Evaluation of Tigecycline Susceptibility Testing Methods for Expanded-Spectrum Cephalosporin- and Carbapenem-Resistant Gram-Negative Pathogens

ABSTRACT We evaluated the Vitek2, Etest, and MIC Test Strip (MTS) methods of tigecycline susceptibility testing with 241 expanded-spectrum cephalosporin-resistant and/or carbapenem-resistant Enterobacteriaceae and Acinetobacter baumannii clinical isolates by using dry-form broth microdilution (BMD) as the reference method. The MIC50/90s were as follows: BMD, 1/4 μg/ml; Vitek2, 4/≥8 μg/ml; Etest, 2/4 μg/ml; MTS, 0.5/2 μg/ml. Vitek2 produced 9.1/21.2% major errors, Etest produced 0.4/0.8% major errors, and MTS produced no major errors but 0.4/3.3% very major errors (FDA/EUCAST breakpoints). Vitek2 tigecycline results require confirmation by BMD or Etest for multidrug-resistant pathogens.

Linezolid Dependence in Staphylococcus epidermidis Bloodstream Isolates

We document linezolid dependence among 5 highly linezolid-resistant (LRSE) Staphylococcus epidermidis bloodstream isolates that grew substantially faster at 32 µg/mL linezolid presence. These isolates carried the mutations T2504A and C2534T in multiple 23S rRNA copies and 2 mutations leading to relevant amino acid substitutions in L3 protein. Linezolid dependence could account for increasing LRSE emergence.

Susceptibility patterns to extended-spectrum cephalosporins among Enterobacteriaceae harbouring extended-spectrum β-lactamases using the updated Clinical and Laboratory Standards Institute interpretive criteria

We examined the effect of applying the updated 2010 Clinical and Laboratory Standards Institute (CLSI) susceptibility breakpoints for extended-spectrum cephalosporins (ESCs) to detect extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. In total, 202 ESBL-producing, plasmidic AmpC- and carbapenemase-negative isolates derived from separate patients were collected from three Greek hospitals during 2007-2011, including 150 Escherichia coli, 43 Klebsiella pneumoniae and 9 Enterobacter cloacae clinical isolates. ESBLs were detected using the ESBL CLSI confirmatory test and PCR assays. Sequencing analysis showed that 91 (45.0%) of the ESBL-producers carried the bla(CTX-M-3) gene, 66 (32.7%) carried the bla(CTX-M-15) gene and the remaining 45 (22.3%) carried the bla(SHV-5) gene. Minimum inhibitory concentrations for cefotaxime, ceftazidime and cefepime were determined by the agar dilution method. Based on the new CLSI breakpoints, 13 (6.4%) of the ESBL-producers were susceptible to cefotaxime, 90 (44.6%) to ceftazidime and 112 (55.4%) to cefepime; as many as 145 (71.8%) were susceptible to at least one ESC. Among the 150 E. coli, 12 (8.0%), 87 (58.0%) and 79 (52.7%) were susceptible to cefotaxime, ceftazidime and cefepime, respectively, whilst among the 43 K. pneumoniae, 1 (2.3%), 3 (7.0%) and 25 (58.1%) were susceptible to the above ESCs, respectively. None of the nine E. cloacae were susceptible to cefotaxime and ceftazidime, but all except one were susceptible to cefepime. By implementation of the new 2010 CLSI breakpoints, a considerable proportion of ESBL-possessing Enterobacteriaceae would be reported as susceptible, mostly to ceftazidime and cefepime, leading to possible infection control and therapeutic implications.

Wide dissemination of linezolid-resistant Staphylococcus epidermidis in Greece is associated with a linezolid-dependent ST22 clone

OBJECTIVES Dependence on linezolid was recently described as significant growth acceleration of linezolid-resistant Staphylococcus epidermidis (LRSE) isolates upon linezolid exposure. We investigated the possible contribution of linezolid dependence to LRSE dissemination in Greece. METHODS Linezolid resistance rates were estimated in six tertiary hospitals located throughout Greece between 2011 and 2013. Sixty-three randomly selected LRSE recovered in these hospitals during this period were studied. Growth curve analysis was conducted with and without linezolid. Clonality of the isolates was investigated by PFGE and MLST. RESULTS During the study period, the LRSE rate in the participating hospitals rose significantly from 6.9% to 9% (P = 0.006); the increase was more prominent in ICUs (from 15.1% to 20.9%; P = 0.005). Forty-seven (74.6%) of the 63 LRSE, derived from all study hospitals, clearly exhibited linezolid dependence, growing significantly faster in the presence of 16 and 32 mg/L linezolid. Of note, 61 (96.8%) LRSE exhibited a single macrorestriction pattern and belonged to ST22, which included all linezolid-dependent LRSE. The remaining two LRSE belonged to unique STs. Five of six linezolid-dependent isolates tested also exhibited linezolid dependence upon exposure to 8 mg/L linezolid. Interestingly, five of six ST22 linezolid-non-dependent isolates tested developed linezolid dependence when linezolid exposure preceded growth analysis. CONCLUSIONS The rapid LRSE dissemination in Greek hospitals threatens linezolid activity. The observation that most LRSE belonged to ST22 and expressed dependence on linezolid clearly implies that the spread of linezolid resistance should have been driven by this trait, which provided the LRSE with a selective advantage under linezolid pressure.

Predominance of international clone 2 OXA-23-producing-Acinetobacter baumannii clinical isolates in Greece, 2015: results of a nationwide study ★

In a previous nationwide study in Greece, OXA-58 was the sole carbapenemase present among carbapenem-resistant Acinetobacter baumannii (CRAB) isolated between 2000 and 2009. In this study, the antibiotic resistances, carbapenemase gene content and clonal relatedness of 194 single-patient CRAB clinical isolates collected randomly during 2015 from 11 tertiary hospitals located throughout Greece were investigated. Antimicrobial susceptibility was determined using commercial and dilution methods. PCR assays for carbapenemase genes were performed. Clonality was tested by a scheme based on two multiplex PCRs and single-locus blaOXA-51-like sequence-based typing. Furthermore, Pasteurs multilocus sequence typing (MLST) scheme and pulsed-field gel electrophoresis (PFGE) were applied to 31 selected representative isolates. The most active antibiotics were trimethoprim/sulfamethoxazole (SXT) (34.6% of isolates susceptible), minocycline (71.6%), colistin (72.7%) and tigecycline (MIC50/90 values, 1/2 mg/L). The blaOXA-23-like gene was identified in 188 isolates (96.9%), blaOXA-23-like together with blaOXA-58-like in 3 isolates (1.5%), blaOXA-58-like in 2 isolates (1.0%) and blaOXA-40-like in 1 isolate (0.5%). ISAba1 was found upstream of the blaOXA-23-like gene in all isolates. International clone (IC) 2 comprised 157 isolates (80.9%), IC1 comprised 36 isolates (18.6%) and ST78 comprised 1 isolate (0.5%). All IC2 and IC1 isolates tested by MLST were ST2 and ST1, respectively. Seven PFGE types were detected. IC2 isolates were resistant to more antibiotics than IC1, except for SXT. This nationwide study showed that CRAB isolates in Greek hospitals currently produce almost uniformly the OXA-23 carbapenemase and belong mainly to IC2 and, to a lesser extent, IC1. Of particular concern, colistin susceptibility is recently severely reduced.

First-year results of an antibiotic stewardship program in a Greek tertiary care hospital

The aim of this study was to investigate the effect of the implementation of an antibiotic stewardship program (ASP) on antibiotic consumption in our 428-bed hospital. The Infection Control Committee implemented an ASP beginning in January 2016, aiming to reduce inappropriate antibiotic use through improved prescribing practices. The ASP included both pre-authorization and prospective audit and feedback strategies. We collected pharmacy and hospital data for the years 2015 (pre-intervention) and 2016 (post-intervention). Consumption data were expressed as daily defined doses (DDDs) per 100 patient-days (PD) and the significance of the differences between 2015 and 2016 was assessed by paired t-test. Antibiotic resistance rates for the most important hospital pathogens were monitored for 2015–2016. The ASP effectively reduced consumption of most antimicrobials; total antibiotic use decreased by 16.7% (from 104.3 in 2015 to 86.9 DDDs/100 patient-days in 2016, p < 0.001) owing to reduction of 19.1% for non-restricted and 13.8% for restricted antibiotics. Important restricted antimicrobials, such as colistin, carbapenems, quinolones and tigecycline showed significantly decreased usage post-intervention. Significant changes in the resistance rates were not observed, except a decreasing trend for colistin and tigecycline (Acinetobacter baumannii and Klebsiella pneumoniae) and also vancomycin (enterococci). The ASP was successful in terms of reducing the antibiotic consumption for the first year of its implementation. Interestingly, antimicrobials requiring pre-authorization exhibited a lower reduction than other antibiotics. Potential effects of the ASP in reducing resistance rates remain to be shown.