Emergence of OXA-162 Carbapenemase- and DHA-1 AmpC Cephalosporinase-Producing Sequence Type 11 Klebsiella pneumoniae Causing Community-Onset Infection in Greece.

OXA-48-like carbapenemases have only recently emerged in Europe. OXA-162 is a rare OXA-48 variant usually coexpressed with extended-spectrum ß-lactamases. Here, we report the identification of the first OXA-162 carbapenemase-producing Klebsiella pneumoniae isolates, 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 Enterobacteriaceae strain.

The Balkan region: NDM-1-producing Klebsiella pneumoniae ST11 clonal strain causing outbreaks in Greece.

OBJECTIVES: Despite the fact that the NDM-1 carbapenemase has successfully disseminated worldwide, outbreaks remain uncommon in the European region. We describe the characteristics of the first outbreaks caused by NDM-1-producing Klebsiella pneumoniae clonal isolates in Greece. METHODS: Between January 2010 and June 2013, 132 non-repetitive carbapenem-resistant Enterobacteriaceae isolates, which gave a positive modified Hodge test and were phenotypically suspected of metallo-ß-lactamase production, were recovered from patients hospitalized at Ioannina University Hospital. Resistance genes were identified by PCR and sequencing. Plasmid profiling, conjugation experiments, enterobacterial repetitive intergenic consensus PCR, PFGE and multilocus sequence typing (MLST) were performed. Patient records were retrieved to access patterns of acquisition. RESULTS: Molecular testing verified the presence in 78 K. pneumoniae isolates, collected from 71 patients, of the blaNDM-1 gene. The blaCTX-M-15, blaOXA-1 and blaTEM-1 genes were also present in most isolates. The blaNDM-1 gene was located on a narrow host range IncFII-type plasmid, of ∼95 kb, flanked upstream by a non-truncated ISAba125 element and downstream by the bleMBL gene. Genotyping clustered all K. pneumoniae isolates into a single clonal type with one subtype and MLST assigned them to sequence type 11. Two outbreaks were noted, the first between November and December 2011 involving four patients and the second initiated in May 2012 and ongoing, involving the remaining patients. All but two cases were characterized as hospital acquired. No links to immigration or travel history to endemic areas were established. CONCLUSIONS: This survey highlights the successful undetected dissemination of yet another carbapenemase in Greece and strengthens the hypothesis of a latent NDM-1 cluster in the Balkan region.

Outbreak caused by an ertapenem-resistant, CTX-M-15-producing Klebsiella pneumoniae sequence type 101 clone carrying an OmpK36 porin variant.

Although 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. pneumoniae strain expressing an OmpK36 porin variant. From May 2012 to November 2012, 37 ertapenem-resistant K. pneumoniae isolates 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-15 gene on a conjugative 100-kb plasmid, disruption in the expression of the ompK35 gene, 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. pneumoniae isolates 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.

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-ß-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.

Imported Klebsiella pneumoniae carbapenemase-producing K. pneumoniae clones in a Greek hospital: impact of infection control measures for restraining their dissemination.

The recent emergence of carbapenemase-producing Enterobacteriaceae strains represents a major threat for hospitalized patients. We document the dissemination and control of carbapenemase-producing Klebsiella pneumoniae clones in a Greek hospital. During a 3-year study period (January 2009 to December 2011), carbapenemase-producing K. pneumoniae strains were isolated from clinical samples from 73 individual patients. Phenotyping and molecular testing confirmed that 52 patients were infected with K. pneumoniae carbapenemase 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. pneumoniae patient 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. pneumoniae were 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.

Comparative evaluation of a prototype chromogenic medium (ChromID CARBA) for detecting carbapenemase-producing Enterobacteriaceae in surveillance rectal swabs.

Carbapenemase-producing Enterobacteriaceae (CPE) are an increasing problem worldwide, and rectal swab surveillance is recommended as a component of infection control programs. The performance of a prototype chromogenic medium (chromID CARBA) was evaluated and compared with media tested by four other screening methods: (i) overnight selective enrichment in 5 ml tryptic soy broth with a 10-µg ertapenem disk followed by plating onto MacConkey agar (CDC-TS), (ii) short selective enrichment in 9 ml brain heart infusion broth with a 10-µg ertapenem disk followed by plating onto chromID ESBL medium (ESBL-BH), (iii) direct plating onto chromID ESBL, and (iv) direct plating onto MacConkey agar supplemented with meropenem (1 µg/ml) (MCM). The screening methods were applied to detect CPE in 200 rectal swab specimens taken from different hospitalized patients. Identification and antimicrobial susceptibility were performed by the Vitek 2 system. Carbapenem MICs were checked by Etest. Carbapenemase production was confirmed using the modified Hodge test, combined-disk tests, and PCR assays. In total, 133 presumptive CPE strains were detected. Phenotypic and genotypic assays confirmed 92 strains to be CPE (56 KPC-positive Klebsiella pneumoniae, 29 VIM-positive K. pneumoniae, and 7 KPC-positive Enterobacter aerogenes strains) recovered from 73 patients, while the remaining 41 strains were confirmed to be CPE negative (19 ESBL producers and 22 nonfermenters). chromID CARBA, ESBL-BH, and chromID ESBL exhibited the highest sensitivity (92.4%), followed by CDC-TS and MCM (89.1%) (P = 0.631). The specificity was greater for chromID CARBA (96.9%) and ESBL-BH (93.2%) than for CDC-TS (86.4%), MCM (85.2%), and chromID ESBL (84.7%) (P = 0.014). In conclusion, chromID CARBA was found to be a rapid and accurate culture screening method for active CPE surveillance.

Detection of Pseudomonas aeruginosa isolates of the international clonal complex 11 carrying the blaPER-1 extended-spectrum ß-lactamase gene in Greece.

OBJECTIVES: The extended-spectrum ß-lactamase (ESBL) PER-1 initially disseminated among Pseudomonas aeruginosa strains in Turkey. Despite reports from other European countries, such strains have not been detected in Greece until now. We describe the first bla(PER-1)-positive P. aeruginosa isolates from Greece and their genetic environment. METHODS: From January 2008 to December 2009, 287 consecutive non-duplicate P. aeruginosa isolates with reduced susceptibility or resistance to ceftazidime (MIC >8 mg/L) were screened for ESBL production with a modified boronic acid-based double-disc synergy test. Phenotypically ESBL-positive isolates were subjected to agar dilution, PFGE and multilocus sequence typing (MLST). Broad-spectrum bla genes were identified by PCR and sequencing. Plasmid analysis and conjugation experiments were performed. The location of the bla(PER-1) gene was detected by Southern blotting and its genetic environment was characterized using inverse PCR. RESULTS: Five isolates were phenotypically positive for ESBL production, exhibited resistance to cefepime, ceftazidime, aztreonam and meropenem, and carried the bla(PER-1) gene. MLST showed that they belonged to sequence type (ST) 235, which belongs to the international clonal complex 11. Four isolates had the same PFGE pattern. Southern blotting revealed the chromosomal location of the bla(PER-1) gene. Analysis of the bla(PER-1) flanking regions showed identity to transposon Tn1213 downstream and 1406 bp upstream of bla(PER-1). Further upstream, an orfA gene and ISPa12 were identified; both were truncated by the insertion of IS6100. CONCLUSIONS: This study confirmed the presence of PER-1-producing P. aeruginosa strains in Greece. The chromosomal location of bla(PER-1), as part of a truncated transposon, suggests clonal expansion rather than horizontal gene transfer.

Containment of an outbreak of KPC-3-producing Klebsiella pneumoniae in Italy.

From March 2009 to May 2009, 24 carbapenem-resistant Klebsiella pneumoniae isolates were recovered from 16 patients hospitalized in an Italian intensive care unit (ICU). All isolates contained KPC-3 carbapenemase and belonged to a single pulsed-field gel electrophoresis (PFGE) clone of multilocus sequence type 258 (designated as ST258). A multimodal infection control program was put into effect, and the spread of the KPC-3-producing K. pneumoniae clone was ultimately controlled without closing the ICU to new admissions. Reinforced infection control measures and strict monitoring of the staff adherence were necessary for the control of the outbreak.

Comparative evaluation of combined-disk tests using different boronic acid compounds for detection of klebsiella pneumoniae carbapenemase-producing enterobacteriaceae clinical isolates.

The accurate phenotypic detection of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae is 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 Enterobacteriaceae isolates. To measure the specificity of the tests, 127 genotypically confirmed KPC-negative Enterobacteriaceae isolates 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.

Evolution of multidrug-resistant Acinetobacter baumannii clonal lineages: a 10 year study in Greece (2000-09).

OBJECTIVES: To analyse the evolution and genetic relatedness of Acinetobacter baumannii clonal lineages in Greece during a 10 year period. METHODS: The study included 94 randomly selected A. baumannii clinical isolates recovered from 2000 to 2009 in eight tertiary Greek hospitals. Carbapenem MICs were determined by agar dilution. PCR was applied for carbapenemase genes. Isolates were typed by PFGE and tri-locus sequence typing (3LST), and 25 were also typed by multilocus sequence typing (MLST) developed by the Institut Pasteur, followed by e-Burst analysis. RESULTS: All isolates were multidrug-resistant (MDR); 54 (57.4%) were non-susceptible to imipenem and/or meropenem. The bla(OXA-58) gene was identified in 51 (94.4%) carbapenem-non-susceptible and 15 (37.5%) carbapenem-susceptible isolates; other carbapenemase genes were not detected. Eight different PFGE types were identified. Sequence typing revealed previously characterized 3LST groups (1, 2, 4 and 5) and MLST types (STs) (1, 2, 15, 45 and 54) and the novel STs 85 (in two distant hospitals) and 86. Eight novel 3LST alleles were identified. Fifty-two (55.3%) isolates were assigned to 3LST group 1 and ST2 or ST45, both corresponding to international clonal complex 2 (CC2). Thirty-one (33.0%) isolates were assigned to 3LST group 2 and ST1 (CC1). From 2000 to 2004 63% of isolates belonged to 3LST group 2, but from 2005 to 2009 87.5% of isolates belonged to 3LST group 1; this shift was accompanied by an increase in carbapenem resistance from 43.5% to 64.6% of isolates. CONCLUSIONS: The emergence of MDR A. baumannii in Greece was associated with CC1 and CC2, which are disseminated worldwide, often harbouring the bla(OXA-58) gene. Novel 3LST alleles and STs were also detected, underlining an evolutionary divergence in Greece.

Risk factors and outcomes associated with acquisition of colistin-resistant KPC-producing Klebsiella pneumoniae: a matched case-control study.

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 beta-lactam/beta-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.

In vivo acquisition of a plasmid-mediated bla(KPC-2) gene among clonal isolates of Serratia marcescens.

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 bla(KPC-2) gene during the hospitalization.

Detection of the new metallo-beta-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.

A simple phenotypic method for the differentiation of metallo-beta-lactamases and class A KPC carbapenemases in Enterobacteriaceae clinical isolates.

BACKGROUND: The increasing frequency of class A KPC enzymes and class B metallo-beta-lactamases (MBLs) among Enterobacteriaceae as well as their possible co-production makes their early differentiation urgent. METHODS: A simple phenotypic algorithm employing three combined-disc tests consisting of meropenem alone and with phenylboronic acid (PBA), EDTA, or both PBA and EDTA was designed for the differentiation of KPC and MBL enzymes. Augmentation of the zone of inhibition by >or=5 mm was considered a positive combined-disc test result. A total of 141 genotypically confirmed carbapenemase-positive Enterobacteriaceae clinical isolates (63 KPC producers, 47 MBL producers, and 31 KPC and MBL producers) with various carbapenem MICs were examined. For comparison, 84 genotypically confirmed carbapenemase-negative Enterobacteriaceae clinical isolates [39 extended-spectrum beta-lactamase (ESBL) producers, 22 AmpC producers, and 23 ESBL and AmpC producers] were also tested. RESULTS: The phenotypic algorithm was able to differentiate MBL from KPC producers as well as to detect the possible co-production of both carbapenemases (positive result only with the combined-disc test using meropenem alone and with both PBA and EDTA). The method detected all KPC or MBL producers (sensitivity 100%) as well as 30 of the KPC and MBL producers (sensitivity 96.8%). All three combined-disc tests were negative for non-carbapenemase-producing isolates, except two ESBL and AmpC producers that gave positive combined-disc tests using meropenem alone and with PBA and both PBA and EDTA (specificity for KPC detection 98.8%). CONCLUSIONS: This phenotypic method is very helpful to detect carbapenemase production and provides a simple algorithm for the differentiation of KPC and MBL enzymes, especially in regions where KPC- and MBL-possessing Enterobacteriaceae are highly prevalent.

Evaluation of the performance of Acuitas® Resistome Test and the Acuitas Lighthouse® software for the detection of ß-lactamase-producing microorganisms.

OBJECTIVES: Given the international spread of multidrug-resistant Gram-negative bacteria the need for prompt and precise characterization of underlying resistance traits has evolved into the cornerstone of infection control strategies. Novel commercial molecular tests enable rapid simultaneous testing for multiple resistance genes. We aimed to evaluate the performance of OpGen's Acuitas® Resistome Test and the Acuitas Lighthouse® software. METHODS: The test is tailored towards detecting 46 ß-lactamase genes (SHV and TEM variants associated with wild-type penicillin resistance, extended-spectrum ß-lactamases [ESBLs], acquired AmpCs and carbapenemases) via a microfluidic polymerase chain reaction (PCR) array. In total 118 isolates part of the collection of the Bacteriology Laboratory of the Hellenic Pasteur Institute, specifically 96 enterobacterial isolates and 21 Acinetobacter baumannii, of divergent origins, with previously characterized ß-lactamase content, were tested. RESULTS: In the enterobacterial group all 69 carbapenemase genes of the KPC, VIM, NDM and OXA-48 types were correctly identified (sensitivity, specificity, positive predictive value [PPV] and negative predictive value [NPV] of 100%). Non-ESBL SHV enzymes, ESBLs (CTX-M, GES, VEB types) and acquired AmpC enzymes were also correctly characterized. Of the 35 SHV-ESBLs harboured, correct identification was possible in 32/35 isolates, with overall sensitivity, specificity, PPV and NPV for the Klebsiella pneumoniae group of 89.29%, 100%, 100% and 91.18%, respectively. For the A. baumannii group the test exhibited an overall sensitivity for carbapenemase detection of 96.55% and 100% PPV. CONCLUSIONS: The OpGen Acuitas Resistome Test is an efficient molecular tool that can identify resistance threats in health care institutions with high diagnostic accuracy and be integrated into targeted surveillance protocols.

Use of boronic acid disk tests to detect extended- spectrum beta-lactamases in clinical isolates of KPC carbapenemase-possessing enterobacteriaceae.

We evaluated boronic acid (BA)-based methods for their ability to detect extended-spectrum beta-lactamases (ESBLs) among clinical isolates of KPC-producing members of the Enterobacteriaceae family. 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 > or =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 Enterobacteriaceae producing KPC carbapenemases.

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.

Performance of the ß LACTA™ test for rapid detection of expanded-spectrum cephalosporin-non-susceptible Enterobacteriaceae.

OBJECTIVES: The aim of this study was to examine the performance of the chromogenic ß LACTA™ test for the rapid detection of expanded-spectrum cephalosporin (ESC) non-susceptibility among Enterobacteriaceae in a region endemic for potent ß-lactamases. METHODS: The ß LACTA™ test was applied prospectively on 235 consecutive Enterobacteriaceae clinical isolates and 163 previously characterised ESC-non-susceptible Enterobacteriaceae producing a range of ß-lactamases. RESULTS: The ß LACTA™ test exhibited excellent sensitivity (96.1%) and specificity (98.5%) for the detection of ESC non-susceptibility in the 235 clinical isolates, which harboured mainly extended-spectrum ß-lactamase (ESBL) and KPC- and NDM-type enzymes. Among the 163 challenged archived isolates, some false-negative or uninterpretable results were detected, mostly among ESC-non-susceptible isolates with AmpC/VIM/OXA-48-like enzymes. CONCLUSION: ß LACTA™ may be effectively applied in regions where ESC non-susceptibility among Enterobacteriaceae is mainly due to ESBL, KPC or NDM ß-lactamases.

Carbapenemase-producing Enterobacteriaceae: now that the storm is finally here, how will timely detection help us fight back?

Acquired carbapenemases of the Ambler Classes A, B and D have gradually disseminated over the past decade among isolates of the Enterobacteriaceae family. Located on highly mobile genetic elements, these enzymes have established their presence in successful bacterial clones, signaling the prelude to a novel endemic era in the hospital setting. In such a context, prompt detection of carbapenemase-producing microorganisms seems warranted, not only for the selection of an appropriate antibiotic regimen, but also for organizing and implementing effective surveillance and infection control measures. However, issues regarding detection, confirmatory testing and characterization persist. Determination of carbapenemase production via susceptibility pattern interpretation may not always be feasible, confirmatory testing remains debatable and precise characterization is a challenge for the clinical laboratory. We aim to review the current situation from a laboratory point of view, highlighting the tools that have been proposed in an effort to help outline reliable screening and detection procedures, with inhibitor-based phenotypic testing as the cornerstone of these strategies, and discuss the necessity for implementing identification of possible producers in the everyday workflow.

Emergence of VIM-1 metallo-ß-lactamase-producing Escherichia coli in a neonatal intensive care unit.

A carbapenem-resistant Escherichia coli isolate was recovered from rectal swab of a 12-day-old female neonate, which was admitted to a Greek neonatal intensive care unit (NICU). Phenotypic testing, polymerase chain reaction assays with sequencing, and plasmid analysis revealed that the isolate harbored a plasmid-mediated bla(VIM-1) metallo-ß-lactamase gene. The appearance of a metallo-ß-lactamase-producing E. coli in NICU is worrisome. Further surveys are needed to determine whether such Enterobacteriaceae may also be spreading in other NICUs.