infections. The spread of CTXM–positive bacteria considerably changes the way we think about treating community-acquired infections and limits the oral antibiotics that may be administered. This finding has major implications for treating children, who should not be given fluoroquinolones and tetracyclines. The observation that different blaCTX-M alleles, located on plasmids of different sizes, were involved in clinical infections caused by distinct E. coli clones implies that CTX-M enzymes may become widespread in the community. A possible association of blaCTX-M genes with insertion sequences like ISEcp1B might have contributed to the enhanced expression and mobilization of blaCTX-M genes among E. coli isolates (7). The apparent dissemination of CTX-M producers could represent a substantial barrier in the treatment of community-acquired infections. Additionally, severely ill patients treated in the outpatient setting may transmit such resistant organisms to hospitalized patients.

To characterize the mechanisms implicated in fluoroquinolone (FQ) and expanded-spectrum cephalosporin (ESC) resistance in three clinical and seven faecal multidrug-resistant (MDR; resistant to at least three antimicrobial classes) Escherichia coli isolates from a dog with atopic dermatitis, also suffering from recurrent otitis, that had already been exposed to prolonged antimicrobial treatment and colonized for a long period.MICs of FQs, ESCs and other antimicrobials were determined by the broth microdilution method. Phenotypic tests (efflux pump inhibition and combination disc tests) and isoelectric focusing were combined with genotypic analyses [PCRs, sequencing, conjugation, S1 nuclease PFGE, PCR-based replicon typing, plasmid multilocus sequence typing (pMLST) and PCR mapping] to characterize the molecular basis of FQ and ESC resistance. Isolates were further characterized by MLST and PFGE.Three otitis and five faecal isolates with enrofloxacin MICs of 32 to > 128 mg/L displayed the GyrA:S83L+D87N/ParC:E62K/ParE:G545D pattern harbouring novel ParC and ParE substitutions, whereas the two remaining faecal isolates were susceptible or borderline resistant single-step mutants (GyrA:S83L pattern) and carried qnrS(1). Efflux pump overexpression also contributed to FQ resistance and the MDR phenotype. The three otitis and five faecal isolates also exhibited cefoxitin/ceftazidime MICs of 32-64 mg/L and harboured bla(CMY-2), adjusted to ISEcp1, on an IncI1/ST65 conjugative plasmid, previously described in Salmonella Heidelberg from poultry. Interestingly, all isolates shared an identical MLST type (ST212), with the otitis isolates showing indistinguishable patterns with the high-level resistant faecal E. coli isolates.The long-term maintenance of FQ- and ESC-resistant clones harbouring topoisomerase mutations and a bla(CMY-2)-IncI1/ST65 plasmid in canine commensal flora after prolonged antimicrobial use may contribute to the dissemination of multidrug resistance.

Background: Vancomycin-resistant Enterococcus faecium (VRE) may cause outbreaks in neonatal intensive care units (NICU). We describe a biphasic VRE outbreak and identify risk factors for VRE acquisition.Methods: After the occurrence of 2 cases of VRE infections in a 44-bed NICU, a bundle of interventions was implemented that included active surveillance cultures for VRE, enhanced infection control measures, and audits on antimicrobial use, from June to December 2008. Analysis was performed using polymerase chain reaction and pulse-field gel electrophoresis techniques. A case-control study was conducted to identify risk factors.Results: Among 253 neonates screened, 101 (39.9%) were found to be colonized with VRE. During the first 9 weeks of the study period, 59 new cases were detected. Molecular analysis showed 1 predominant clone. During weeks 10-12, no new cases of VRE colonization were detected; however, at week 13, just when the outbreak appeared to be over, a second wave occurred, with 42 new cases and multiple clones detected. Multivariate analysis identified administration of antimicrobial therapy for late-onset neonatal sepsis and hospitalization during the first month of this outbreak as significant risk factors for VRE colonization.Conclusion: Both a high prevalence of VRE colonization and antimicrobial use promoted the transmission of VRE during this biphasic outbreak. Adherence to infection control measures and antimicrobial stewardship policies are of utmost importance. Copyright (C) 2013 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Pseudomonas aeruginosa has the potential to achieve resistance to carbapenems via the acquisition of carbapenemase-encoding genes, the downregulation of the OprD porin, the overexpression of efflux systems and the overproduction of cephalosporinases. One hundred and fifty carbapenem-non-susceptible isolates from 2008 to 2010 were screened for carbapenemase production, OprD porin loss, efflux pumps overexpression and inducible AmpC beta-lactamase production. For comparison reasons, the presence of the same mechanisms was also assessed in a previous collection of 30 carbapenem-non-susceptible P. aeruginosa isolated between 2003 and 2005. Results showed the accumulation of various resistance mechanisms among VIM-2 producers isolated between 2008 and 2010 with a parallel considerable increase in imipenem MIC90 and the geometric mean of the MIC values of imipenem and meropenem between the two study groups. The accumulation of carbapenem resistance mechanisms highlights the potential of this formidable pathogen for evolutionary success under antibiotic selective pressure.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen associated with a range of nosocomial infections. This microorganism is noted for its intrinsic resistance to antibiotics and for its ability to acquire genes encoding resistance determinants. Among the beta-lactam antibiotics, carbapenems with antipseudomonal activity are important agents for the therapy of infections due to P. aeruginosa. The development of carbapenem resistance among P. aeruginosa strains is multifactorial. Plasmid or integron-mediated carbapenemases, increased expression of efflux systems, reduced porin expression and increased chromosomal cephalosporinase activity have all been defined as contributory factors. Phenotypic tests and molecular techniques are used for the characterization of the resistance determinants. The isolation of carbapenem resistant strains is alarming and requires the implementation of strict infection control measures in order to prevent the spread of carbapenemase encoding genes to unrelated clones or to other bacterial species.

We present a polyclonal outbreak of vancomycin-resistant enterococci (VRE) colonization in a pediatric oncology department and the role of a bundle of actions. After the occurrence of VRE bloodstream infections in 2 patients, an active surveillance of VRE colonization was started. Enhanced infection control measures and closure of the department to new admissions for the first 3 months were implemented. Among 32 patients screened for VRE, 21 were found colonized. Daily prevalence of VRE colonization among hospitalized patients ranged from 40% to 75%, but no new VRE infections occurred. Monthly incidence of VRE colonization decreased from 2.5 to 0.6 cases per 100 occupied bed-days at the end of this outbreak by the implementation of the above-mentioned measures. All VRE isolates tested were Enterococcus faecium carrying VanA gene. Pulsed field gel electrophoresis showed a polyclonal outbreak. A case-control study did not show any particular risk factors for colonization. High use of glycopeptide was noted before study outbreak that was drastically decreased during the study but only temporarily. Control of VRE in pediatric oncology departments with high colonization rates is challenging and requires a multifaceted strategy. Polyclonal spread of VRE found in this study suggests a possible effect of prior antimicrobial overuse and the critical need for antimicrobial stewardship especially in the era of multidrug-resistant bacteria.

Department of Clinical Microbiology, Hippokration Hospital, Thessaloniki, Greece; Department of Microbiology, Medical School, University of Thessaly, Larissa, Greece; Laboratory of Parasitology, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece; Department of Pediatric Surgery, Hippokration Hospital, Thessaloniki, Greece; Third Department of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece

This study investigated the prevalence of vancomycin-resistant enterococci (VRE) in the broiler production environment after the avoparcin ban and their epidemiological relationship with human clinical VRE from the same geographical regions in Greece.Caecal contents from broilers (n500) from eight livestock farms and faecal samples from poultry slaughterers (n50), all collected in two slaughterhouses during 200508, were analysed for species and vancomycin resistance gene identification using multiplex PCR. Sixty-three human clinical vancomycin-resistant Enterococcus faecium (VREF) isolates, obtained during 200609, were also examined. Discriminant analysis (DA) was used to establish the relationship of antimicrobial resistance profiles (ARPs) among broiler, poultry slaughterer and human clinical VREF. PFGE was conducted to study the genetic relatedness among VREF from the different sources.A total of 120 VRE were recovered from 113 (22.6) broiler samples. VREF carrying the vanA gene were predominant, being recovered from 72 (14.4) samples from five (62.5) broiler farms. Concerning poultry slaughterers, VREF were recovered from 10 (20) samples. Susceptibility testing revealed that broiler VREF were consistently resistant to tetracycline, whereas 93.7 of clinical VREF were resistant to ampicillin. Furthermore, 92.1 of clinical VREF compared with 54.4 of broiler VREF were multiresistant (resistant to at least five antimicrobial classes). DA classified broiler and human clinical VREF into their corresponding source with high classification rates (100 and 85.7, respectively), while the classification rate of poultry slaughterer VREF was relatively low (50), with 40 of them classified closely to broiler VREF. PFGE patterns were clearly related to the source of the VREF, with broiler isolates being clustered distinctly from all human isolates.A remarkable persistence of VREF was observed in the broiler production environment even 10 years after the avoparcin ban. Human and broiler VREF belonged to clearly unrelated populations, strongly indicating no clonal spread of VREF among the different sources, even between broilers and poultry slaughterers, despite them sharing common ARPs, as also supported by DA.

We report 570 carbapenemase-producing Klebsiella pneumoniae (CPKP) clinical isolates in a 1,040-bed Greek tertiary hospital during 2004 to 2010. The first CPKP (VIM-producing) was isolated in September 2004. Despite initial containment, VIM producers have become endemic since 2006. KPC-producing K. pneumoniae was first isolated in August 2007 from a patient who came from Israel, spread rapidly, and outcompeted VIM. Overall, 267 (47%) VIM-producing and 301 (53%) KPC-producing strains were isolated, including 141 (24.7%) from patients with bacteraemia. Two isolates carrying both VIM and KPC were isolated in two consecutive months in 2009, but not since. The prevalence of CPKP increased from 0% in 2003 to 38.3% in 2010 (p<0.0001). All genotyped KPC producers harboured bla KPC-2 and belonged to two clones, among which the hyperepidemic Greek clone, related to those from the United States and Israel, predominated. Most metallo-beta-lactamase (MBL) producers carried the bla VIM-1 gene and belonged to several clones, whereas all but one isolate with bla VIM-12 were clustered within a five-month period, arising from one clone. Resistance to non-beta-lactam antibiotics was also increased among CPKP. They were almost invariably resistant to ciprofloxacin and trimethoprim-sulfamethoxazole. Resistance to colistin increased from 3.5% (4/115) in 2008 to 20.8% (25/120) in 2010, and resistance to tigecycline also increased. Following reinforcement of infection control measures, prevalence of CPKP (mainly KPC) has been reduced since mid-2009 (from 46% in 2009 to 38.3% in 2010). In view of the exhaustion of available therapies, investment in infection control resources and optimal antibiotic use is urgently required.

AIM:The aim of this preliminary study was to investigate the in vitro effect of "non-antibiotic" trimebutine against reference strains Staphylococcus aureus ATCC 29213, ATCC 25923, Escherichia coli ATCC 25922, ATCC 35218, Pseudomonas aeruginosa ATCC 27853 and Enterococcus faecalis ATCC 29212; microbiota that are potentially involved in the pathophysiology of post-infectious functional gastrointestinal disorders.METHODS:Trimebutine activity was assessed by the broth microdilution method according to Clinical and Laboratory Standards Institute recommendations against reference strains S. aureus ATCC 29213 and ATCC 25923, E. coli ATCC 25922 and ATCC 35218, P. aeruginosa ATCC 27853 and E. faecalis ATCC 29212. Bactericidal activity of the compound was determined by spreading a 10 μL aliquot on Mueller-Hinton agar from each dilution showing non-visible growth. All tests were carried out in triplicate.RESULTS:Trimebutine was active against all strains tested presenting with MIC ranging from 1024 to 4000 mg/L. MIC and MBC were similar for E. coli ATCC 25922 and P. aeruginosa ATCC 27853 whereas for Gram-positive isolates and E. coli ATCC 35218 the MBC was higher.CONCLUSIONS:We demonstrated the in vitro bacteriostatic/bactericidal activity of trimebutine against bacteria frequently colonizing the gastrointestinal tract and potentially involved in human gastrointestinal infections that might trigger post-infectious functional gastrointestinal disorders.

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.

The outcome of patients with urinary tract infections caused by extended spectrum β-lactamases (ESBL)-producing bacteria (cases) was compared with that of matched controls with urinary tract infections caused by non-extended spectrum β-lactamases-producing isolates. Significantly, more case patients received inappropriate empiric therapy than controls. Nevertheless, clinical and microbiologic outcomes as well as formation of renal scars did not differ between the 2 groups.

A Klebsiella pneumoniae clinical isolate resistant to imipenem was recovered from a wound sample. The patient, a 57-year-old man, underwent a surgical resection of small bowel and sigmoid colon and was treated with multiple courses of antimicrobials. PCR analysis revealed that the clinical isolate was carrying simultaneously bla(VIM-1), bla(KPC-2), bla(SHV) and bla(TEM) genes. The concomitant presence of these genes is alarming and poses therapeutic as well as infection control problems.

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 blaKPC-2 gene, and 17 of 18 MBL-producing isolates carried blaVIM-1 Acute Physiology and Chronic Health Evaluation II score (odds ratio, 1.13 [95% confidence interval, 1.03–1.25]; P = .02) 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 (P = .04) and in-hospital death (P = .03) 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.

We describe a hospital outbreak caused by colistin-resistant Klebsiella pneumoniae producing KPC-2 beta-lactamase in two distinct medical centres. Seven clinical isolates of K. pneumoniae exhibiting resistance to carbapenems were collected from patients with hospital-acquired infection. All isolates were phenotypically positive for carbapenemase activity but negative for metallo-beta-lactamase production. PCR analysis using specific primers for bla(KPC), bla(SHV), bla(TEM) and bla(CTX-M) demonstrated that all clinical strains of K. pneumoniae from hospital A and one isolate from hospital B were genetically related and carried bla(KPC-2) in addition to bla(SHV-12). In contrast, the remaining isolate carried bla(S)(HV-5) with bla(K)(PC-2) and yielded a different profile. These results indicate the clonal spread of KPC producers between hospitals as well as the acquisition of KPC genes by different K. pneumoniae strains. All isolates were resistant to carbapenems, beta-lactams, ciprofloxacin, aminoglycosides and colistin, but intermediately susceptible to tigecycline and susceptible to gentamicin. The infection was fatal in five cases. The emergence of colistin-resistant K. pneumoniae possessing bla(KPC)(-2) underscores the implementation of strict control measures to prevent their dissemination of these organisms in hospitals.

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.

We analysed trends in antimicrobial resistance of Enterococcus faecalis (N=1498) and E. faecium (N=625) recovered from clinical infections during 2002-2007 in a Greek tertiary care hospital. Molecular assays were used to confirm speciation and genotype of vancomycin-resistant enterococci (VRE). The incidence of infections per 1000 admissions caused by E. faecalis and E. faecium increased during the study period (chi(2) for trend=25.5 and 13.3, respectively; P<0.0001). Resistance to ampicillin, high level gentamicin and streptomycin, vancomycin, teicoplanin and linezolid was found in E. faecalis/E. faecium at rates of 1.3/82.4%, 45.6/51.2%, 48.9/69.1%, 0.5/9.6%, 0.1/8.2% and 0.3/1.6%, respectively. The vanA gene was identified in 79.1% of the VRE isolates, with vanB found in the remaining 20.1%. Analysis of antimicrobial resistance trends showed consistently high rates of ampicillin resistance among E. faecium isolates. For both enterococcal species, high level resistance to gentamicin and streptomycin were noted to have increased significantly (P<0.0001). Regardless of these alarming trends, strains exhibiting resistance to oxazolidinones seem to be only sporadic in our region and a trend toward increasing resistance rates to glycopeptides was not detected.

ABSTRACT A study was designed to investigate the molecular epidemiological characteristics of multidrug-resistant outbreak-related Pseudomonas aeruginosa isolates collected in a university hospital in northern Greece. Of 29 nonreplicate P. aeruginosa isolates resistant to carbapenems and ceftazidime, 14 were positive for metallo-β-lactamase production. PCR analyses with primers specific for bla VIM and bla IMP revealed that 13 isolates carried a novel bla VIM-2 gene variant, designated bla VIM-17 , and only 1 isolate carried bla VIM-2 , a gene predominant among P. aeruginosa strains in Greek hospitals. Pulsed-field gel electrophoresis of XbaI-digested genomic DNAs showed a close genetic relationship for 12 of 13 bla VIM-17 -carrying outbreak-related isolates, which were of the O11 serotype; the clonally unrelated isolate carrying bla VIM-17 was of the O12 serotype. PCR mapping strategies for the detection of class 1 integrons and sequencing approaches revealed the presence of integrons containing one bla VIM cassette flanked by two aacA29 cassettes. These integrons were similar but not identical to In59 (GenBank accession number AF263519) initially described in France. All isolates carrying bla VIM-17 , regardless of their genetic profile, had an identical integron, named In59.3, indicating that although the hospital outbreak was mainly due to clonal dissemination, the horizontal transmission of the bla VIM-17 -containing integron among P. aeruginosa isolates should also have occurred. An outbreak-related isolate and a control strain, both of which carried the bla VIM-2 gene but which were clonally distinct, had an identical integron, named In59.2, which differed only at the level of the bla VIM gene from In59.3 integrons, suggesting a common ancestry. The spread of the bla VIM-17 -containing integron in clonally unrelated P. aeruginosa isolates without any evidence of plasmid carriage is probably associated with a transposon.