Michalis Polemis

An update of the evolving epidemic of blaKPC-2-carrying Klebsiella pneumoniae in Greece (2009–10)

OBJECTIVES To follow the epidemic of KPC-2-producing Klebsiella pneumoniae in Greece. METHODS KPC-2-producing isolates (n = 378) were collected during January 2009-April 2010 in 40 Greek hospitals. bla(KPC) and bla(VIM) were detected by PCR. Carbapenemase production was confirmed by spectrophotometry. Sequences flanking bla(KPC-2) and their plasmid carriers were studied. Isolates were typed by PFGE and multilocus sequence typing (MLST). RESULTS All 378 isolates were bla(KPC-2) positive; 18 also carried bla(VIM-1/VIM-4). Higher isolation frequencies were observed in Athens and Crete. Isolates were classified into 13 PFGE types and 11 sequence types (STs). ST258 was predominant (n = 322), followed by ST147 (n = 20), ST383 (n = 9), ST133 (n = 6), ST274 (n = 4) and ST323 (n = 3). Of the remaining isolates, seven were distributed into five STs (11, 17, 340 and the novel 494 and 495) and seven were not typed. bla(KPC-2) could not be transferred from ST258 isolates, in contrast to isolates of ST17, ST133, ST147, ST274, ST494 and ST495. All bla(KPC-2)-encoding plasmids were of similar size (∼100 kb) and showed indistinguishable restriction fragment length polymorphism (RFLP) patterns except those from the ST340 isolates. Sequences flanking bla(KPC-2) revealed that the Tn4401a isoform was present in plasmids from all STs except ST340 containing Tn4401b. Co-production of VIM enzymes was observed in isolates of ST147, ST323 and ST383. CONCLUSIONS Apart from the epidemic of KPC-2-producing K. pneumoniae belonging to ST258 in Greece, diffusion of bla(KPC-2) to at least 10 additional STs has taken place. Notably, strains from three of the latter STs (147, 323 and 383) were found to carry both bla(KPC-2) and bla(VIM).

Emerging Klebsiella pneumoniae Isolates Coproducing KPC-2 and VIM-1 Carbapenemases

VIM-1-producing Klebsiella pneumoniae strains, increasingly isolated in Greece since 2002, constitute the majority of multiresistant clinical isolates of this species found in most Greek hospitals ([9][1]). Moreover, following reports on Klebsiella pneumoniae carbapenemase (KPC)-producing isolates

Carbapenem-resistant Klebsiella pneumoniae infections in a Greek intensive care unit: Molecular characterisation and treatment challenges

Acquisition of carbapenemase-producing Klebsiella pneumoniae (CP-Kp) strains poses a major threat to critically ill patients. The objectives of this study were to describe the epidemiology of CP-Kp isolates as well as the clinical outcome associated with the corresponding infections and to identify risk factors for mortality of intensive care unit (ICU) patients in a Greek hospital. A prospective, observational study was conducted in a nine-bed general ICU over a 2-year period (April 2010-March 2012). Imipenem-resistant K. pneumoniae isolates recovered from clinical samples of ICU patients were prospectively collected and studied for the presence of carbapenemases. Isolates were submitted to molecular typing using pulsed-field gel electrophoresis (PFGE). In total, 61 CP-Kp isolates (48 KPC-producers and 13 VIM-producers) were recovered from 58 ICU patients. The majority of KPC-producers were classified into a single PFGE type, indicating potent clonal dissemination. Among the 32 infected patients, bacteraemia was diagnosed in 16. Tigecycline+colistin was the most common combination antimicrobial regimen. Infection-attributable mortality was 43.8%. Regarding mortality risk factors, non-survivors were older (P=0.080), all of them presented with septic shock (P=0.010) and they had higher Sepsis-related Organ Failure Assessment (SOFA) scores at infection onset (P=0.004) compared with survivors. Appropriate definitive treatment and combination regimens were not associated with patient survival. In conclusion, CP-Kp infections are associated with limited treatment options and high in-hospital mortality. Effective measures for preventing dissemination of respective isolates in the hospital setting are required.

Supplementation of growth media with Zn2+ facilitates detection of VIM-2-producing Pseudomonas aeruginosa.

Isolation rates of Pseudomonas aeruginosa producing zinc-dependent class B metallo-β-lactamases (MBLs) mainly of the VIM and IMP types are increasing worldwide (7). These enzymes exhibit wide hydrolysis spectra, including carbapenems, and are strongly inhibited by chelating agents such as EDTA. Based on the latter property, various MBL-detecting assays have been developed (1). EDTA-imipenem synergy tests are widely utilized in hospitals in Greece, where the incidence of VIM-positive P. aeruginosa is considered among the highest in Europe (www.rivm.nl/earss). However, observations in various hospitals in Athens and the reference laboratory of the National School of Public Health (NSPH) indicate suboptimal sensitivity of the EDTA-based methods. In this study, we attempted to evaluate the effect of zinc supplementation of the test medium on the performance of these methods. Forty-three P. aeruginosa isolates submitted for testing in the NSPH from 13 hospitals during 2006 due to difficulties in interpreting the results of the EDTA-imipenem synergy methods were included in the study. MICs of β-lactams were determined by the Etest (AB Biodisk, Solna, Sweden). PCR assays for detection of MBL genes were performed as described previously (5, 6). The identities of MBL genes were confirmed by sequencing of the respective amplicons. Phenotypic detection of MBLs was performed in Mueller-Hinton agar (MHA) as well as in the same medium in which 70 mg/liter ZnSO4·7H2O had been incorporated (Zn2+ at a final concentration of 250 μM) as suggested by Lee et al. (4). Imipenem-EDTA synergy was assessed with the MBL-Etest (with a ≥8-fold decrease in the MIC of imipenem in the presence of EDTA considered a positive result) as well as two in-house techniques: the double-disk synergy test (DDST), using imipenem (10 μg) and EDTA disks (930 μg) in a 20-mm center-to-center distance, and the combination disk test (CDT), using an imipenem (10 μg) disk alone and containing 930 μg EDTA (with a ≥7-mm increase in inhibition zone considered a positive result) (3). The effects of the Zn2+ supplementation on the Etest MICs of imipenem and ceftazidime were also determined. Twenty-seven (63%) of the 43 isolates carried blaVIM-2 (group A). The remaining 16 isolates (37%) were negative for MBL genes (group B). Imipenem MICs for group A isolates ranged from 1 to >32 μg/ml. The respective range for group B isolates was 4 to 32 μg/ml. Imipenem MICs were in good agreement with those reported by the hospital laboratories. Sensitivity problems (false negatives) were noticed with all three EDTA-based methods employed. The higher sensitivity score was observed with DDST followed by MBL-Etest and CDT. Also four of the group B isolates appeared false positive (Table ​(Table1),1), producing slight although reproducible synergy images. Imipenem MICs of the false-positive isolates ranged from 8 to 32 μg/ml. TABLE 1. Properties of 43 P. aeruginosa isolates Incorporation of Zn2+ in the growth medium resulted in a significant increase in the sensitivity of all three MBL detection methods without compromising specificity. More specifically, in Zn2+-supplemented MHA, the MBL-Etest and CDT correctly identified 27 (sensitivity 100%) and 26 (sensitivity 96%) group A isolates, respectively, while performance of the conventional testing techniques was poor. Likewise, zinc supplementation increased the number of group A isolates characterized as MBL positive by the DDST from 12 to 18, thus improving sensitivity from 44 to 67% (Table ​(Table1).1). A plausible explanation for the positive effect of Zn2+ on MBL detection in P. aeruginosa is that Zn2+ may facilitate formation of functional MBL molecules in the periplasmic space. Also, the relatively high Zn2+ concentrations during growth reduce expression of P. aeruginosa porins and consequently carbapenem diffusion rates (2), further enhancing the effects of carbapenemase activity. This explanation is compatible with the increase in the apparent resistance levels to imipenem and ceftazidime that was more pronounced among VIM-2 producers (Table ​(Table11). Twenty-seven of the 43 submitted P. aeruginosa isolates (15 group A and 12 group B) were readily and correctly characterized in the NSPH by at least one conventional EDTA-based phenotypic method, likely suggesting technical problems in the hospital laboratories. Nevertheless, in a number of isolates, MBL production was not apparent. Despite the limitations of this preliminary study (a relatively small number of VIM-producing isolates), our findings suggest that Zn2+ supplementation may be a useful adjunct for MBL detection in P. aeruginosa and warrants further investigation.

Circulation of a multiresistant, conjugative, IncA/C plasmid within the nosocomial Providencia stuartii population in the Athens area

The objective of the study is to report a multidrug-resistant outbreak of Providencia stuartii that occurred in inpatients in the Athens area in 2012 resulting from a very successful transmissible A/C multidrug-resistant plasmid. Thirteen multidrug-resistant P. stuartii clinical isolates from 5 hospitals were studied. Molecular typing was performed by pulsed-field gel electrophoresis. Antibiotic resistance genes and their genetic surround were detected by PCR and sequencing. Plasmid analysis included conjugation experiments using liquid cultures, sizing by S1 digestion, and incompatibility replicon typing by PCR. Isolates were grouped into 2 distinct clonal types A and B, exhibiting similarity less than 70%. Isolates of type A were recovered from patients hospitalized in 4 different hospitals with no obvious epidemiological linkage, while isolates of type B were recovered from patients treated in a single hospital. Both clonal types harbored a conjugative plasmid of 130 bp and IncA/C replicon type carrying 5 β-lactamase genes bla(SHV-5), bla(VEB-1), bla(VIM-1), bla(OXA-10), and bla(TEM-1) and aminoglycosides resistant determinants. All β-lactamase genes were included in stable structures as IS26, IS1999, and In-e541. The current plasmid seemed to have many common determinants with previously reported plasmids derived from P. stuartii and Proteus mirabilis clinical isolates and exhibited the ability to circulate in nosocomial bacterial populations.

Outbreak of pan-susceptible Klebsiella pneumoniae in a neonatal intensive care unit

Abstract We describe the outbreak of a pan-susceptible Klebsiella pneumoniae strain in a neonatal intensive care unit. A total of 7 neonates developed bacteraemia (37% attack rate), of whom 3 died (43% case fatality rate). A birth weight < 1500 g was the only statistically significant risk factor. Despite an extensive environmental investigation, the source was not identified.

An outbreak of a possibly new Salmonella enterica subspecies enterica serovar with the antigenic formula 11:z41:e,n,z15, Greece, March to May 2016: preliminary results

Eleven Salmonella spp. isolates with the antigenic type 11:z41:e,n,z15 - not referred to in the 9th edition of the White-Kauffman-Le Minor Scheme - were identified at the National Reference Laboratory for Salmonella in Greece. Their pulsed-field gel electrophoresis profiles were indistinguishable. No apparent epidemiological link has yet been identified; the results of a case-case study are pending.

Predictions in antibiotics resistance and nosocomial infections monitoring

Nosocomial infections and antibiotic resistance are regarded as critical issues both in clinical medicine as well as in Public health, thus understanding their epidemiology is a priority in the health sector. Our research aims at demonstrating that data mining techniques, such as regression, classification and association rules and assist in discovering interesting patterns in the epidemiological trends of antibiotic resistance in Greek Hospitals. In this work, we present a novel framework which integrates data from multiple hospitals and discovers association rules stored in a data warehouse. Furthermore, this data warehouse is used as a source for extracting interesting and valid predictions by applying techniques such as regression and classification. Our system is fully operational and treats real-world data from the WHONET, a software installed on the majority of Greek member hospitals of the ”Greek System for Surveillance of Antimicrobial Resistance” network. The contributions of the proposed framework are i. a standardized workflow for the seamless integration of data produced in various hospitals into a consistent data warehouse and b. the use of a mechanisms to predict hidden future behavior on large datasets, using regression and classification algorithms, which can provide significant surveillance warnings.