Danai Sofianou

A simple phenotypic method for the differentiation of metallo-β-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 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.

Bloodstream infections caused by metallo-β-lactamase/Klebsiella pneumoniae carbapenemase-producing K. pneumoniae among intensive care unit patients in Greece: risk factors for infection and impact of type of resistance on outcomes.

OBJECTIVE To determine risk factors for bloodstream infections (BSIs) caused by Klebsiella pneumoniae producing metallo-β-lactamases (MBLs) or K. pneumoniae carbapenemases (KPCs), as well as risk factors for mortality associated with carbapenem-resistant K. pneumoniae, among intensive care unit (ICU) patients. METHODS Two case-control studies were conducted in a patient cohort with K. pneumoniae BSIs in an 8-bed ICU in a Greek hospital from January 1, 2007, through December 31, 2008. In study 1, patients with K. pneumoniae BSIs were allocated among 3 groups according to isolate susceptibility profile: (1) carbapenem-susceptible insolates (control group), (2) MBL-producing isolates, or (3) KPC-producing isolates. The MBL and KPC groups were compared with the control group to identify risk factors for development of K. pneumoniae BSI. In study 2, patients with K. pneumoniae BSIs who died were compared with survivors to identify risk factors for mortality. RESULTS Fifty-nine patients had K. pneumoniae BSIs (22 with carbapenem-susceptible isolates, 18 with MBL-producing isolates, and 19 with KPC-producing isolates). All KPC-producing isolates carried the bla(KPC-2) gene, and 17 of 18 MBL-producing isolates carried bla(VIM-1). Acute Physiology and Chronic Health Evaluation II score (odds ratio, 1.13 [95% confidence interval, 1.03-1.25]; [Formula: see text]) was independently associated with KPC-producing K. pneumoniae BSIs. Nine (41%) of 22 control patients, 8 (44%) of 18 MBL group patients, and 13 (68%) of 19 KPC group patients died in the ICU. Nine (41%) of 22 control patients, 10 (56%) of 18 MBL group patients, and 15 (79%) of 19 KPC group patients died in the hospital. Isolation of KPC-producing K. pneumoniae was an independent predictor of ICU death ([Formula: see text]) and in-hospital death ([Formula: see text]) but not infection-attributable death. CONCLUSIONS BSIs due to KPC-producing K. pneumoniae resulted in significantly increased mortality. The accurate and rapid detection of these pathogens is necessary for therapeutic considerations and for the implementation of infection control measures to contain them.

Colistin heteroresistance in carbapenemase-producing Klebsiella pneumoniae

Sir, Klebsiella pneumoniae is an important pathogen that causes severe and life-threatening infections. Most isolates demonstrate resistance to various antimicrobial agents. Carbapenems have been widely used to treat serious infections with multidrug-resistant K. pneumoniae. However, the increasing use of these compounds has led to the emergence of carbapenemresistant isolates via acquired genes encoding carbapenemhydrolysing enzymes that inactivate carbapenems. Colistin was among the last-resort therapy for treating infections caused by these pathogens. In the present study we report on colistin heteroresistance in carbapenemase-producing apparently colistin-susceptible K. pneumoniae isolates recovered from patients with and without prior exposure to this drug. A total of 20 K. pneumoniae clinical isolates harbouring carbapenem-hydrolysing enzymes that were classified as colistin-susceptible by routine susceptibility testing were consecutively collected between March and August 2009 from separate patients with nosocomial infections hospitalized in several wards of our hospital. Eight isolates were obtained from patients who had a previous treatment with colistin for more than 3 days, whereas 12 isolates were from patients who did not receive colistin. The identification of isolates and initial susceptibility testing were performed by the VITEK 2 automated system (bioMerieux, Marcy l’Etoile, France). Phenotypic and molecular techniques demonstrated that 13 isolates were KPC producers whereas 7 were VIM producers. The MICs of colistin were determined by the broth microdilution method according to CLSI recommendations and interpreted using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for Enterobacteriaceae (susceptible≤2 mg/L; resistant.2 mg/L). K. pneumoniae isolates were assessed for heteroresistance by spreading a 50 mL aliquot from a 24 h culture serially diluted in saline to a turbidity equivalent to that of a 0.4–0.5 McFarland standard (approximately 10 cfu/mL) on Mueller–Hinton agar plates containing 0, 0.5, 1, 2, 3, 4, 5, 6 and 8 mg/L colistin sulphate. The plates were incubated at 358C for 48 h and colonies were counted. The frequency of resistant subpopulations at the highest drug concentration was calculated by dividing the number of colonies grown on an antibioticcontaining plate by the colony counts from the same bacterial inoculum plated onto antibiotic-free plates. The MIC for isolates grown at the highest colistin concentration was reassessed after serial daily subcultures on antibiotic-free medium for 2 weeks in order to evaluate whether this resistance was stable. Macrorestriction profiles of K. pneumoniae isolates were investigated by PFGE. Sixteen of the study strains were colistin susceptible (MIC≤2 mg/L) while four strains (4, 5, 7 and 13) were shown to be resistant by broth microdilution (colistin MIC 4 mg/L) and were not studied further for heteroresistance. Results of population analysis profiles for colistin are reported in Table 1. Four strains (11, 14, 15 and 16), derived from patients without colistin treatment, did not exhibit any heteroresistance (no growth in 2 mg/L colistin in population analyses). In population analyses, 12 strains yielded heteroresistant subpopulations that grew in up to 8 mg/L colistin. The proportion of these subpopulations was 3.5×10 to 4.4×10 for isolates exposed to colistin and 1.5×10 to 3.2×10 for isolates not exposed to colistin. When heteroresistant colonies obtained from population analyses were retested for colistin MICs after daily passages on colistin-free medium, all strains demonstrated MICs.8 mg/L except one (strain 8, Table 1). The MIC for this strain, which was recovered from a patient who had previously been treated with colistin, initially increased from 1 mg/L to 4 mg/L, however, after subculture of the resistant colonies onto antibiotic-free medium, its MIC decreased to the susceptible range (1 mg/L). PFGE showed heterogeneity among the study strains that were classified into five distinct clones; two clones among VIM-producing isolates, two clones among KPC-producing isolates and one clone that comprised two KPC producers and one VIM producer (Table 1). Colistin resistance among carbapenemase-producing K. pneumoniae is increasing in Greece, and stable heteroresistance might play a role in resistance development. In fact, in our hospital, resistance rates rose from 0% in 2007 to 8.13% in 2008 and to 24.3% in 2009. Colistinand carbapenem-resistant isolates have been involved in hospital outbreaks with high mortality rates. Furthermore, the increasing reports of colistin-susceptible isolates harbouring resistant subpopulations are of great concern. Commercial automated systems may fail to detect most heteroresistant isolates, and Etest results depend on the medium used. As this species constitutes the majority of carbapenemase-producing Enterobacteriaceae isolated from clinical specimens worldwide, it is very important to develop a reliable and easy-to-use susceptibility testing method to detect resistant subpopulations.

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.

VIM-1 Metallo-β-lactamase in Acinetobacter baumannii

In 2004 and 2005, 5 metallo-β-lactamase (MBL)-positive Acinetobacter baumannii isolates were found in 2 Greek hospitals. Isolates were unrelated and carried blaVIM-1 in a class 1 integron; blaOXA-51- and blaOXA-58-like carbapenemase genes were also detected. VIM-1 MBL in Acinetobacter spp. causes concern, given the increasing resistance of this species.

Candida tropicalis in a Neonatal Intensive Care Unit: Epidemiologic and Molecular Analysis of an Outbreak of Infection with an Uncommon Neonatal Pathogen

ABSTRACT From June to July 1998, two episodes of Candida tropicalis fungemia occurred in the Aristotle University neonatal intensive care unit (ICU). To investigate this uncommon event, a prospective study of fungal colonization and infection was conducted. From December 1998 to December 1999, surveillance cultures of the oral cavities and perinea of the 593 of the 781 neonates admitted to the neonatal ICU who were expected to stay for >7 days were performed. Potential environmental reservoirs and possible risk factors for acquisition of C. tropicalis were searched for. Molecular epidemiologic studies by two methods of restriction fragment length polymorphism analysis and two methods of random amplified polymorphic DNA analysis were performed. Seventy-two neonates were colonized by yeasts (12.1%), of which 30 were colonized by Candida albicans, 17 were colonized by C. tropicalis, and 5 were colonized by Candida parapsilosis. From December 1998 to December 1999, 10 cases of fungemia occurred; 6 were due to C. parapsilosis, 2 were due to C. tropicalis, 1 was due to Candida glabrata, and 1 was due to Trichosporon asahii (12.8/1,000 admissions). Fungemia occurred more frequently in colonized than in noncolonized neonates (P < 0.0001). Genetic analysis of 11 colonization isolates and the two late blood isolates of C. tropicalis demonstrated two genotypes. One blood isolate and nine colonization isolates belonged to a single type. The fungemia/colonization ratio of C. parapsilosis (3/5) was greater than that of C. tropicalis (2/17, P = 0.05), other non-C. albicans Candida spp. (1/11, P = 0.02), or C. albicans (0/27, P = 0.05). Extensive environmental cultures revealed no common source of C. tropicalis or C. parapsilosis. There was neither prophylactic use of azoles nor other risk factors found for acquisition of C. tropicalis except for total parenteral nutrition. A substantial risk of colonization by non-C. albicans Candida spp. in the neonatal ICU may lead to a preponderance of C. tropicalis as a significant cause of neonatal fungemia.

Fungal Peritonitis Complicating Peritoneal Dialysis during an 11-Year Period: Report of 46 Cases

The incidence of fungal peritonitis (FP) and the fungi that caused FP were evaluated in 422 patients treated with peritoneal dialysis. During an 11-year period, 804 episodes of peritonitis occurred, 46 (5.7%) of which were caused by fungi. Treatment was successful for 39 patients. Early diagnosis of FP and prompt therapy decreases morbidity and mortality.

Outbreak of Infections Caused by Enterobacter cloacae Producing the Integron-Associated β-Lactamase IBC-1 in a Neonatal Intensive Care Unit of a Greek Hospital

ABSTRACT Nineteen of 27 ceftazidime-resistant Enterobacter cloacae isolates from a neonatal intensive care unit in Thessaloniki, Greece, had genes coding for the novel extended-spectrum β-lactamase IBC-1; 18 of those 19 harbored similar conjugative plasmids and belonged to two distinct genetic lineages. A synergy test with ceftazidime and imipenem enabled us to identify five unrelated blaIBC-1-carrying E. cloacae isolates from other wards of the hospital. It seems that this integron-associated gene is capable of dispersing both by clonal spread and by gene dissemination.