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Background Staphylococcus aureus bacteremia (SAB) disproportionately affects Black patients. The reasons for this disparity are unclear. Methods We evaluated a prospectively ascertained cohort of patients with SAB from 1995 to 2020. Clinical characteristics, bacterial genotypes, and outcome were compared among Black and White patients with SAB. Multivariable logistic regression models were used to determine factors independently associated with the outcomes. Results Among 3068 patients with SAB, 1107 (36%) were Black. Black patients were younger (median, 56 years vs 63 years; P < .001) and had higher rates of diabetes (47.5% vs 34.5%, P < .001), hemodialysis dependence (40.0% vs 7.3%, P < .001), and human immunodeficiency virus (6.4% vs 0.6%, P < .001). Black patients had higher rates of methicillin-resistant S. aureus (49.3% vs 44.9%, P = .020), including the USA300 hypervirulent clone (11.5% vs 8.4%, P = .007). White patients had higher rates of corticosteroid use (22.4% vs 15.8%, P < .0001) and surgery in the preceding 30 days (28.1% vs 18.7%, P < .001). Although the median Acute Physiology Score (APS) at the time of initial SAB diagnosis was significantly higher in Black patients (median APS, 9; interquartile range [IQR], 5–14 vs median APS, 7; IQR, 4–12; P < .001), race was not associated with 90-day mortality (risk ratio, 1.02; 95% confidence interval, .93–1.12), and rates of metastatic infection were lower among Black patients (37.2% vs 41.3% White, P = .029). Conclusions Despite differences in Black patients' higher APS on presentation and more risk factors, including a 5 times higher risk of hemodialysis dependence, 90-day mortality among Black and White patients with SAB was similar.

The aim of this study was to characterize the 16S rRNA methylase (RMT) genes in aminoglycoside-resistant Enterobacterales and Pseudomonas aeruginosa isolates in 2015-2016 in hospitals in Athens, Greece. Single-patient, Gram-negative clinical isolates resistant to both amikacin and gentamicin (n = 292) were consecutively collected during a two-year period (2015-2016) in five tertiary care hospitals in Athens. RMT genes were detected by PCR. In all RMT-producing isolates, ESBL and carbapenemase production was confirmed by PCR, and the clonal relatedness and the plasmid contents were also characterized. None of the 138 P. aeruginosa isolates harbored any of the RMT genes surveyed although some were highly resistant to aminoglycosides (MICs > = 512 mg/L). Among 154 Enterobacterales, 31 Providencia stuartii (93.9%), 42 Klebsiella pneumoniae (37.8%), six Proteus mirabilis (75%), and two Escherichia coli (100%) isolates were confirmed as highly resistant to amikacin, gentamicin, and tobramycin with MICs ≥ 512 mg/L, harboring mainly the rmtB (98.8%). All were carbapenemase producers. P. stuartii, P. mirabilis, and E. coli produced VIM-type carbapenemases. K. pneumoniae produced KPC- (n = 34, 81.0%), OXA-48 (n = 4, 9.5%), KPC- and VIM- (n = 3, 7.1%), or only VIM-type (n = 1, 2.4%) enzymes. Two groups of similar IncC plasmids were detected one harboring rmtB1, blaVEB-1, blaOXA-10, and blaTEM-1, and the other additionally blaVIM-1 and blaSHV-5. Among RMT-producing Enterobacterales, rmtB1 predominated and was associated with carbapenemase-encoding gene(s). Similar IncC plasmids carrying a multiresistant region, including ESBL genes, and in the case of VIM-producing isolates, the blaVIM-1, were responsible for this dissemination. The co-dissemination of these genes poses a public health threat.

Background To understand the clinical, bacterial, and host characteristics associated with recurrent Staphylococcus aureus bacteremia (R-SAB), patients with R-SAB were compared to contemporaneous patients with a single episode of SAB (S-SAB). Methods All SAB isolates underwent spa genotyping. All isolates from R-SAB patients underwent pulsed-field gel electrophoresis (PFGE). PFGE-indistinguishable pairs from 40 patients underwent whole genome sequencing (WGS). Acute phase plasma from R-SAB and S-SAB patients was matched 1:1 for age, race, sex, and bacterial genotype, and underwent cytokine quantification using 25-analyte multiplex bead array. Results R-SAB occurred in 69 (9.1%) of the 756 study patients. Of the 69 patients, 30 experienced relapse (43.5%) and 39 reinfection (56.5%). Age, race, hemodialysis dependence, presence of foreign body, methicillin-resistant Staphyloccus aureus, and persistent bacteremia were individually associated with likelihood of recurrence. Multivariate risk modeling revealed that black hemodialysis patients were nearly 2 times more likely (odds ratio [OR] = 9.652 [95% confidence interval [CI], 5.402–17.418]) than white hemodialysis patients (OR = 4.53 [95% CI, 1.696–10.879]) to experience R-SAB. WGS confirmed PFGE interpretations in all cases. Median RANTES (regulated on activation, normal T cell expressed and secreted) levels in acute phase plasma from the initial episode of SAB were higher in R-SAB than in matched S-SAB controls (P = .0053, false discovery rate Conclusion This study identified several risk factors for R-SAB. The largest risk for R-SAB is among black hemodialysis patients. Higher RANTES levels in R-SAB compared to matched controls warrants further study.

The aim of this study was to study the molecular epidemiology of 16S rRNA-methylase (RMT)-producing clinical Acinetobacter baumannii isolates from hospitals in Athens, Greece. Single-patient A. bau...

Background We conducted a longitudinal study to evaluate changes in the clinical presentation and epidemiology of Staphylococcus aureus bacteremia (SAB) in an academic, US medical center. Methods Consecutive patients with monomicrobial SAB were enrolled from January 1995 to December 2015. Each person’s initial bloodstream S. aureus isolate was genotyped using spa typing. Clonal complexes (CCs) were assigned using Ridom StaphType software. Changes over time in both the patient and bacterial characteristics were estimated with linear regression. Associations between genotypes or clinical characteristics and complications were estimated using multivariable regression models. Results Among the 2348 eligible participants, 54.2% had an implantable, foreign body of some type. This proportion increased significantly during the 21-year study period, by 0.96% annually (P = .002), as did comorbid conditions and acquisition outside of the hospital. Rates of any metastatic complication also significantly increased, by 0.94% annually (P = .019). Among the corresponding bloodstream S. aureus isolates, spa-CC012 (multi-locus sequence type [MLST] CC30), -CC004 (MLST CC45), -CC189 (MLST CC1), and -CC084 (MLST CC15) all significantly declined during the study period, while spa-CC008 (MLST CC8) significantly increased. Patients with SAB due to spa-CC008 were significantly more likely to develop metastatic complications in general, and abscesses, septic emboli, and persistent bacteremia in particular. After adjusting for demographic, racial, and clinical variables, the USA300 variant of spa-CC008 was independently associated with metastatic complications (odds ratio 1.42; 95% confidence interval 1.02–1.99). Conclusions Systematic approaches for monitoring complications of SAB and genotyping the corresponding bloodstream isolates will help identify the emergence of hypervirulent clones and likely improve clinical management of this syndrome.

Objectives Colistin is often the last option to treat infections caused by multidrug-resistant micro-organisms such as carbapenem-resistant Klebsiella pneumoniae. Antimicrobial susceptibility testing of colistin has been fraught with difficulties, which resulted in the need for updated recommendations from the CLSI and EUCAST. Both committees proposed that the ISO 20776-1-2016 standard broth microdilution (BMD) method must be the preferred method for colistin minimum inhibitory concentration (MIC) testing. The objective of this study was to evaluate the commercial ComASP™ Colistin (Liofilchem® srl), a compact panel containing the antibiotic in seven two-fold dilutions (0.25–16 mg/L), using frozen BMD plates (according to CLSI) as reference. Methods Colistin susceptibility testing was performed on a nationwide collection of 392 carbapenem-resistant K. pneumoniae isolates using BMD according to the CLSI and by ComASP™ Colistin according to the manufacturer’s recommendations. Susceptibility test results were interpreted according to EUCAST v.7.1 breakpoints (2017). Colistin was active in vitro against 251 K. pneumoniae isolates (64.0%) with an MIC range of 0.5 mg/L to >64 mg/L and MIC50/90 values of 1/64 mg/L. Essential and categorical agreements were calculated according to ISO20776-2. Results ComASP™ Colistin showed high levels of overall/evaluable essential agreement (94.9%/93.6%) and categorical agreement (97.2%), with very major errors in 0.7% (1/141 colistin-resistant), and met the current acceptance criteria proposed by the CLSI. Ten major errors were observed [4.0% (10/251) colistin-susceptible], three of which were within essential agreement. Conclusions ComASP™ Colistin is a commercial BMD method that reliably determined colistin MICs in a large collection of carbapenem-resistant K. pneumoniae isolates.

In December 2019, the Antibacterial Resistance Leadership Group (ARLG) was awarded funding for another 7-year cycle to support a clinical research network on antibacterial resistance. ARLG 2.0 has 3 overarching research priorities: infections caused by antibiotic-resistant (AR) gram-negative bacteria, infections caused by AR gram-positive bacteria, and diagnostic tests to optimize use of antibiotics. To support the next generation of AR researchers, the ARLG offers 3 mentoring opportunities: the ARLG Fellowship, Early Stage Investigator seed grants, and the Trialists in Training Program. The purpose of this article is to update the scientific community on the progress made in the original funding period and to encourage submission of clinical research that addresses 1 or more of the research priority areas of ARLG 2.0.

Three ceftazidime-avibactam-resistant KPC-2-producing Klebsiella pneumoniae strains of ST39 were isolated in Greece, from rectal swabs of three patients after 10–15 days of treatment. The patients were treated with ceftazidime-avibactam as monotherapy or in combination with colistin. Two of these strains harbored a D179Y or a D179V substitution in the Ω loop of KPC-2, corresponding to KPC-33, or to the novel KPC-57, respectively. The third strain had a 15 amino acid insertion after position 259 in the KPC-2, corresponding to KPC-44.

ObjectivesWe evaluated the in vitro activity of ceftolozane/tazobactam and comparator agents against MDR non-MBL Pseudomonas aeruginosa isolates collected from nine Greek hospitals and we assessed the potential synergistic interaction between ceftolozane/tazobactam and amikacin.MethodsA total of 160 non-MBL P. aeruginosa isolates collected in 2016 were tested for susceptibility to ceftolozane/tazobactam and seven comparator agents including ceftazidime/avibactam. Time–kill assays were performed for synergy testing using ceftolozane/tazobactam 60 or 7.5 mg/L, corresponding to the peak and trough concentrations of a 1.5 g q8h dose, respectively, in combination with 69 mg/L amikacin, corresponding to the free peak plasma concentration. Synergy was defined as a ≥2 log10 cfu/mL reduction compared with the most active agent.ResultsOverall, ceftolozane/tazobactam inhibited 64.4% of the P. aeruginosa strains at ≤4 mg/L. Colistin was the most active agent (MIC50/90, 0.5/2 mg/L; 96.3% susceptible) followed by ceftazidime/avibactam (MIC50/90, 4/16 mg/L; 80.6% susceptible). GES-type enzymes were predominantly responsible for ceftolozane/tazobactam resistance; 81.6% of the non-producers were susceptible. MICs for the P. aeruginosa isolates selected for synergy testing were 2–32 mg/L ceftolozane/tazobactam and 2–128 mg/L amikacin. The combination of ceftolozane/tazobactam with amikacin was synergistic against 85.0% of all the isolates tested and against 75.0% of the GES producers. No antagonistic interactions were observed.ConclusionsCeftolozane/tazobactam demonstrated good in vitro activity against MDR/XDR P. aeruginosa clinical isolates, including strains with co-resistance to other antipseudomonal drugs. In combination with amikacin, a synergistic interaction at 24 h was observed against 85.0% of P. aeruginosa strains tested, including isolates with ceftolozane/tazobactam MICs of 32 mg/L or GES producers.

Purpose. The aim of this study was to evaluate the in vitro activity of double-carbapenem combinations against OXA-48-producing Klebsiella pneumoniae clinical isolates. Methodology. Double combinations of ertapenem, meropenem and imipenem were evaluated for synergy and bactericidal activity using the time–kill methodology. All antibiotics were tested at 10 mg l−1 and at a sub-inhibitory concentration of 0.5× minimum inhibitory concentration (MIC) for isolates with a carbapenem MIC≤8 mg l−1. Synergy was defined as a ≥2log10 colony-forming units (c.f.u.) ml−1 decrease of viable colonies at 24 h compared to the most active carbapenem alone. Results. Ten distinct K. pneumoniae clinical isolates were tested. All carried bla OXA-48 and bla CTX-M-15, and exhibited an MIC range of 64–128, 4–32 and 1–32 mg l−1 for ertapenem, meropenem and imipenem, respectively. Out of 48 isolate-combinations, synergy was observed in 9 (18.8 %) and cidal activity was observed in 13 (27.1 %). In vitro synergistic activity was noted for 5 out of 29 (17.2 %) ertapenem-, 6 out of 29 (20.7 %) meropenem- and 7 out of 38 (18.4 %) imipenem-containing combinations. No combination exhibited antagonism. Bactericidal activity was observed in 7 (24.1 %) ertapenem-, 8 (27.6 %) meropenem- and 11 (28.9 %) imipenem-containing combinations. Among the sub-inhibitory concentration combinations, three (15 %) ertapenem-, four (20 %) meropenem- and three (15 %) imipenem-containing ones showed synergistic interaction. Conclusion. Dual combinations of carbapenems, including those containing sub-inhibitory concentrations of antibiotics, were synergistic against multidrug-resistant (MDR) and extensively drug-resistant (XDR) K. pneumoniae isolates harbouring bla OXA-48.

Background. There is little information about the efficacy of active alternative drugs to carbapenems except beta-lactam/beta-lactamase inhibitors for the treatment of bloodstream infections (BSIs) due to extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E). The objective of this study was to assess the outcomes of patients with BSI due to ESBL-E who received empiric therapy with such drugs (other active drugs [OADs]) or carbapenems. Methods. A multinational retrospective cohort study of patients with BSI due to ESBL-E who received empiric treatment with OADs or carbapenems was performed. Cox regression including a propensity score for receiving OADs was performed to analyze 30-day all-cause mortality as main outcome. Clinical failure and length of stay were also analyzed. Results. Overall, 335 patients were included; 249 received empiric carbapenems and 86 OADs. The most frequent OADs were aminoglycosides (43 patients) and fluoroquinolones (20 patients). Empiric therapy with OADs was not associated with mortality (hazard ratio [HR], 0.75; 95% confidence interval [CI],.38-1.48) in the Cox regression analysis. Propensity score-matched pairs, subgroups, and sensitivity analyses did not show different trends; specifically, the adjusted HR for aminoglycosides was 1.05 (95% CI,.51-2.16). OADs were neither associated with 14-day clinical failure (adjusted odds ratio, 0.62; 95% CI,.29-1.36) nor length of hospital stay. Conclusions. We were unable to show that empiric treatment with OAD was associated with a worse outcome compared with carbapenems. This information allows more options to be considered for empiric therapy, at least for some patients, depending on local susceptibility patterns of ESBL-E.

OBJECTIVETo evaluate the efficacy of copper-coating in reducing environmental colonization in an intensive-care unit (ICU) with multidrug-resistant-organism (MDRO) endemicityDESIGNInterventional, comparative crossover trialSETTINGThe general ICU of Attikon University hospital in Athens, GreecePATIENTSThose admitted to ICU compartments A and B during the study periodMETHODSBefore any intervention (phase 1), the optimum sampling method using 2 nylon swabs was validated. In phase 2, 6 copper-coated beds (ie, with coated upper, lower, and side rails) and accessories (ie, coated side table, intravenous [i.v.] pole stands, side-cart handles, and manual antiseptic dispenser cover) were introduced as follows: During phase 2a (September 2011 to February 2012), coated items were placed next to noncoated ones (controls) in both compartments A and B; during phase 2b (May 2012 to January 2013), all copper-coated items were placed in compartment A, and all noncoated ones (controls) in compartment B. Patients were randomly assigned to available beds. Environmental samples were cultured quantitatively for clinically important bacteria. Clinical and demographic data were collected from medical records.RESULTSCopper coating significantly reduced the percentage of colonized surfaces (55.6% vs 72.5%; PP=.003) or by enterococci (4% vs 17%; P=.014), the total bioburden (2,858 vs 7,631 cfu/100 cm2; P=.008), and the bioburden of gram-negative isolates, specifically (261 vs 1,266 cfu/100 cm2; P=.049). This effect was more pronounced when the ratio of coated surfaces around the patient was increased (phase 2b).CONCLUSIONSCopper-coated items in an ICU setting with endemic high antimicrobial resistance reduced environmental colonization by MDROs.Infect Control Hosp Epidemiol 2017;38:765–771

The publisher regrets that the article has been published online on 01 March 2019 with errors in Table 1. In the originally published Table 1, the percentage of Imipenem-relebactam susceptibility was incorrectly written as 8 0, while correct data is 98.0. Also, in Meropenem row, column MIC

Relebactam is a β-lactamase inhibitor of class A and class C β-lactamases, including carbapenemases. We evaluated the ability of relebactam to restore imipenem susceptibility against a collection of Klebsiella pneumoniae isolates from Greek hospitals. We tested 314 non-MBL carbapenemase-producing K. pneumoniae consecutive clinical strains isolated from unique patients at 18 hospitals in Greece, between November 2014 and December 2016. Susceptibility testing of imipenem, imipenem-relebactam, meropenem, doripenem, gentamicin, and colistin was performed using broth microdilution. Additionally, MICs of ceftazidime-avibactam, fosfomycin, and tigecycline were determined by MIC Test Strips. MICs were interpreted per EUCAST breakpoints. Imipenem-relebactam MICs were interpreted using the breakpoints proposed for imipenem. Carbapenemase genes were detected using PCR. Whole genome sequencing was performed for selected isolates. Imipenem-relebactam inhibited 98.0% of the KPC-producing isolates at ≤ 2 mg/L (MIC50/90, 0.25/1 mg/L) and was considerably more active than imipenem (MIC50/90, 32/> 64 mg/L). Reduced activity of imipenem-relebactam was rarely detected (2%) and was associated with chromosomal factors (ompK35 disruption and/or mutated ompK36). Only ceftazidime-avibactam showed in vitro activity comparable to imipenem-relebactam (99.6% susceptible). Relebactam provided only weak potentiation of imipenem activity against K. pneumoniae with class D OXA-48-like enzymes. Relebactam exhibited strong potential for restoring the in vitro activity of imipenem against KPC-producing K. pneumoniae, lowering the imipenem MIC50 and MIC90 from 32 to 0.25 mg/L, and from > 64 to 1 mg/L, respectively. Production of KPC carbapenemase represents the main cause of carbapenem resistance among K. pneumoniae in Greek hospitals (66.5%), and this carbapenemase appears to be very well inhibited by relebactam.

[Scope] The aim of these guidelines is to provide recommendations for decolonizing regimens targeting multidrug-resistant Gram-negative bacteria (MDR-GNB) carriers in all settings., [Methods] These evidence-based guidelines were produced after a systematic review of published studies on decolonization interventions targeting the following MDR-GNB: third-generation cephalosporin-resistant Enterobacteriaceae (3GCephRE), carbapenem-resistant Enterobacteriaceae (CRE), aminoglycoside-resistant Enterobacteriaceae (AGRE), fluoroquinolone-resistant Enterobacteriaceae (FQRE), extremely drug-resistant Pseudomonas aeruginosa (XDRPA), carbapenem-resistant Acinetobacter baumannii (CRAB), cotrimoxazole-resistant Stenotrophomonas maltophilia (CRSM), colistin-resistant Gram-negative organisms (CoRGNB), and pan-drug-resistant Gram-negative organisms (PDRGNB). The recommendations are grouped by MDR-GNB species. Faecal microbiota transplantation has been discussed separately. Four types of outcomes were evaluated for each target MDR-GNB:(a) microbiological outcomes (carriage and eradication rates) at treatment end and at specific post-treatment time-points; (b) clinical outcomes (attributable and all-cause mortality and infection incidence) at the same time-points and length of hospital stay; (c) epidemiological outcomes (acquisition incidence, transmission and outbreaks); and (d) adverse events of decolonization (including resistance development). The level of evidence for and strength of each recommendation were defined according to the GRADE approach. Consensus of a multidisciplinary expert panel was reached through a nominal-group technique for the final list of recommendations., [Recommendations] The panel does not recommend routine decolonization of 3GCephRE and CRE carriers. Evidence is currently insufficient to provide recommendations for or against any intervention in patients colonized with AGRE, CoRGNB, CRAB, CRSM, FQRE, PDRGNB and XDRPA. On the basis of the limited evidence of increased risk of CRE infections in immunocompromised carriers, the panel suggests designing high-quality prospective clinical studies to assess the risk of CRE infections in immunocompromised patients. These trials should include monitoring of development of resistance to decolonizing agents during treatment using stool cultures and antimicrobial susceptibility results according to the EUCAST clinical breakpoints., JTC and JRB are supported by ‘Plan Nacional de I+D+i 2013-2016’ and ‘Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0001; RD16/0016/0008)’, co-financed by the European Development Regional Fund ‘A way to achieve Europe’, Operative Programme Intelligent Growth 2014-2020.

Purpose There are few data in the literature regarding sepsis or septic shock due to extended-spectrum β-lactamases (ESBL)-producing Enterobacteriaceae (E). The aim of this study was to assess predictors of outcome in septic patients with bloodstream infection (BSI) caused by ESBL-E. Methods Patients with severe sepsis or septic shock and BSI due to ESBL-E were selected from the INCREMENT database. The primary endpoint of the study was the evaluation of predictors of outcome after 30 days from development of severe sepsis or septic shock due to ESBL-E infection. Three cohorts were created for analysis: global, empirical-therapy and targeted-therapy cohorts. Results 367 septic patients were analysed. Overall mortality was 43.9% at 30 days. Escherichia coli (62.4%) and Klebsiella pneumoniae (27.2%) were the most frequent isolates. β-lactam/β-lactamase inhibitor (BLBLI) combinations were the most empirically used drug (43.6%), followed by carbapenems (29.4%). Empirical therapy was active in vitro in 249 (67.8%) patients, and escalation of antibiotic therapy was reported in 287 (78.2%) patients. Cox regression analysis showed that age, Charlson Comorbidity Index, McCabe classification, Pitt bacteremia score, abdominal source of infection and escalation of antibiotic therapy were independently associated with 30-day mortality. No differences in survival were reported in patients treated with BLBLI combinations or carbapenems in empirical or definitive therapy. Conclusions BSI due to ESBL-E in patients who developed severe sepsis or septic shock was associated with high 30-day mortality. Comorbidities, severity scores, source of infection and antibiotic therapy escalation were important determinants of unfavorable outcome.

The aim of this study was to determine the propensities of imipenem to select for resistant Pseudomonas aeruginosa mutants by determining the mutant prevention concentrations (MPCs) for 9 unrelated clinical isolates and the accession of any relationship with mechanisms of resistance development. The MPC/MIC ratios ranged from 4 to 16. Detection of resistance mechanisms in the mutant derivatives of the nine isolates mainly revealed inactivating mutations in the gene coding for outer membrane protein OprD. Point mutations leading to premature stop codons or amino acid substitution S278P, ≥1bp deletion leading to frameshift mutations and interruption of the oprD by an insertion sequence, were observed. MPC and mutant selection window (MSW) are unique parameters that may guide the implementation of antimicrobial treatment, providing useful information about the necessary imipenem concentration needed in the infection area, in order to avoid the emergence of resistance, especially in clinical situations with high bacterial load.

Carbapenemase-producing Enterobacteriaceae (CPE) have spread worldwide, causing serious infections with increasing frequency. CPE are resistant to almost all available antibiotics, complicating therapy and limiting treatment options. Mortality rates associated with CPE infections are unacceptably high, indicating that the current therapeutic approaches are inadequate and must be revised. Here, we review 20 clinical studies (including those describing the largest cohorts of CPE-infected patients) that provided the necessary information regarding isolate and patient characteristics and treatment schemes, as well as a clear assessment of outcome. The data summarized here indicate that treatment with a single in vitro active agent resulted in mortality rates not significantly different from that observed in patients treated with no active therapy, whereas combination therapy with two or more in vitro active agents was superior to monotherapy, providing a clear survival benefit (mortality rate, 27.4% vs. 38.7%; p

The in vitro activity of the combination colistin/daptomycin was evaluated against multidrug-resistant Acinetobacter baumannii clinical isolates. Clonal relationships were assessed by pulsed-field gel electrophoresis. The following synergy studies were undertaken: (i) daptomycin MICs were determined by E-test on Mueller-Hinton agar plates supplemented with a subinhibitory concentration of colistin; and (ii) time-kill methodology using tubes containing an inoculum of 5×10(5)CFU/mL and subinhibitory concentrations of each antibiotic alone or in combination subcultured at 0, 5 and 24h for colony counting. Synergy was defined as ≥2log10CFU/mL decrease of viable colonies compared with colistin alone. Ten colistin-susceptible and four colistin-resistant A. baumannii isolates were tested. Isolates were assigned to nine different clonal types. Enhanced in vitro activity of the combination was detected only against colistin-susceptible isolates; using plates supplemented with colistin, the daptomycin MIC was reduced by 4- to 128-fold. From a total of 30 isolate-concentration combinations in time-kill studies, a synergistic interaction was detected in 16 (53.3%). The combination exhibited synergy against 8 and 12 of these combinations at 5h and 24h, respectively. No antagonism was detected. Colistin alone was bactericidal against two colistin-susceptible isolates at 24h, whereas the combination was bactericidal against 9 colistin-susceptible isolates at 24h. Against all colistin-resistant isolates, the combination exhibited a static effect and indifference in time-kill studies. Potent in vitro synergistic interactions between colistin and daptomycin provide evidence that this unorthodox combination may be beneficial in the treatment of colistin-susceptible multidrug-resistant A. baumannii.

We report our experience using the double-carbapenem combination as salvage therapy for patients with untreatable infections caused by KPC-2- producing Klebsiella pneumoniae. A total of 27 patients in two institutions in Athens, Greece suffering from complicated urinary tract infections (16) with or without secondary bacteraemia (four and 12 respectively), primary (six) or catheter-related bloodstream infections (two), HAP or VAP (two) and external ventricular drainage infection (one) were treated exclusively with ertapenem and high-dose prolonged infusion meropenem because in-vitro active antimicrobials were unavailable (19) or failed (four) or were contraindicated (six). Most patients presented with severe infections with median APACHE II score of 17 and 11 of those patients (40.7%) had severe sepsis (five) or septic shock (six). The clinical and microbiological success was 77.8 and 74.1% respectively. Crude mortality was 29.6% with attributable mortality of 11.1%. Adverse events, none of them severe, were reported in four patients (14.8%). The double-carbapenem combination as an exclusive regimen represents a safe and valid salvage therapy for untreatable infections by extensively- or pandrug-resistant KPC-producing K.pneumoniae.

Purpose. The aim of the study was to investigate the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in an unselected collection of bloodstream isolates recovered over an 18-month period in a laboratory affiliated to a university hospital in Athens, Greece, and to assess their impact on the in vitro activity of ciprofloxacin and levofloxacin. Methods. Eight PMQR genes were screened by PCR and sequencing. All PMQR-positive isolates were submitted to isoelectric focusing for β-lactamase detection, conjugation or transformation, time-kill assays, mutant prevention concentrationand inoculum effect evaluation. PCR and sequencing of gyrA and parC were performed for detection of chromosomal mutations. Results. Among 96 Gram-negative isolates, 7 (7.3 %) carried one or more PMQR genes. qnrS1 was the most prevalent (5.2 %), followed by aac(6′ )-Ib-cr (4.2 %) and their combination (2 %). Cloning was successful for three isolates. The presence of a single PMQR determinant without any target modification was not associated with quinolone resistance with one exception, S tenotrophomonas maltophilia carrying qnrS1, which was resistant to norfloxacin and ciprofloxacin, but in this isolate, additional mechanisms of quinolone resistance cannot be excluded. All PMQR-positive isolates showed a significant inoculum effect. The mutant prevention concentrations of ciprofloxacin against the quinolone-susceptible clinical isolates ranged from 0.38 to 32 mg l−1 and those of levofloxacin from 1 to 32 mg l−1. Conclusions. PMQRs compromised the bactericidal activity of ciprofloxacin and levofloxacin when expressed in Enterobacter cloacae, S. maltophilia or Klebsiella pneumoniae and when more than one co-existed. PMQR determinants represent an unrecognized threat, capable to compromise the in vitro activity of quinolones if expressed in a favourable genetic environment and to favour selection of resistant mutants by widening the mutant selection window of these agents.

Objectives We characterized the first ceftazidime-avibactam-resistant KPC-producing-Klebsiella pneumoniae clinical isolate detected in Greece, before the introduction of ceftazidime-avibactam in clinical practice. Methods K. pneumoniae KP-90 was isolated from a hospitalized patient in Thessaloniki during a nationwide surveillance study conducted between 2014 and 2016. Antimicrobial susceptibility was tested against a panel of agents. Whole-genome sequencing (Ion Torrent TM platform) of the isolate was carried out to identify the acquired resistance genes and mutations that were associated with ceftazidime-avibactam resistance. Results The K. pneumoniae isolate belonged to multilocus sequence type ST258 and harboured blaKPC-23 as the only carbapenemase gene. The isolate had a minimum inhibitory concentration (MIC) of 16 mg/L to ceftazidime-avibactam and was highly resistant to imipenem, meropenem (MICs, 512 mg/L) and ceftazidime (MIC, >1024 mg/L). blaKPC-23 was detected on a Tn4401a transposon, located on a pKPQIL-type plasmid. A non-functional outer membrane protein OmpK35 and an OmpK36 variant that had been previously associated with K. pneumoniae isolates of ST258 were detected. Transformation studies with Escherichia coli TOP10 showed that KPC-23 offered similar carbapenem MICs as KPC-2 and KPC-3. However, KPC-23 conferred a four-fold higher ceftazidime MIC (>1024 mg/L), which in the presence of avibactam was reduced (>7-fold) to 8 mg/L, which is just within the limit of the susceptibility breakpoint. Conclusions Ceftazidime-avibactam resistance in a KPC-23- producing K. pneumoniae clinical isolate was due to increased ceftazidime hydrolysis and was likely enhanced by OmpK35 porin deficiency.

The antimicrobial activity of copper surfaces against a variety of contemporary carbapenemase-producing Gram-negative bacteria representative of the most problematic nosocomial pathogens worldwide was evaluated.Twenty-four clinical isolates, comprising four of Escherichia coli, two of Enterobacter spp., eight of Klebsiella pneumoniae and five each of Pseudomonas aeruginosa and Acinetobacter baumannii producing either VIM-1 and/or KPC-2 or VIM-2 or OXA-type carbapenemases, were studied. The antimicrobial activity of 99% copper (Cu99%) and a 63% alloy (Cu63%) was evaluated in comparison with that of stainless steel (SS) and polyvinylchloride (PVC) by incubating ∼10(6) cfu/cm(2) of the tested strains on each surface at room temperature.Copper demonstrated antimicrobial activity against all studied isolates. This effect was observed earlier and was more pronounced for Cu99% than for Cu63%. Cu99% showed a bactericidal effect after2 h for A. baumannii, 3 h for Enterobacter spp., 5 h for K. pneumoniae and 6 h for P. aeruginosa and E. coli. No viable colonies were recovered from five (20.8%) isolates after 3 h and from nine (37.5%) isolates after 5 h of incubation on Cu99%.Copper has significant antimicrobial activity against multidrug-resistant nosocomial Gram-negative pathogens. This supports the hypothesis that replacement of high-contact materials with copper could reduce the high burden of environmental contamination around high-risk patients. However, this strategy should be seen as an adjunctive measure to established cleaning protocols and to good hygiene practices for prevention of hospital-acquired infections.

Dissemination of carbapenemase-producing Klebsiella pneumoniae (CP-Kp) has caused a public health crisis that can be paralleled with that caused by the spread of MRSA. CP-Kps, being multidrug-resistant, mainly affect patients with severe underlying conditions in the acute-healthcare setting. CP-Kps are responsible for a variety of life-threatening infections including bacteremia and pneumonia. The shortage of therapeutic options has forced clinicians to use colistin as well as tigecycline, a novel bacteriostatic agent. Although both drugs are generally active in vitro against CP-Kps, therapeutic failures, especially in bacteremias, are quite common. The authors suggest here, after reviewing the literature, that use of the latter drugs should be re-assessed and optimized. The authors have also summarized experimental and clinical data indicating that exploitation of the pharmacokinetic/pharmacodynamic features of carbapenems may provide solutions in bloodstream infections caused by CP-Kps with low-level resistance to the latter drugs. Most importantly, there is evidence that monotherapy must be avoided.

Purpose To evaluate the in vitro efficacy of several anti-staphylococcal agents against a nationwide collection of contemporary Staphylococcus aureus clinical isolates from several healthcare centres in Greece. Methods Thirty hospitals throughout Greece (18 in Attica) provided all clinical isolates of S.aureus from April 2012 to May 2013 to a central lab to be re-submitted to susceptibility testing. The MICs were evaluated by Vitek® 2 with the exception of ceftaroline (OXOID M.I.C. Evaluator™). Vancomycin and daptomycin MICs were also evaluated by Etest®. Heterogeneously vancomycin-intermediate strains (hVISA) were detected by the Etest® GRD. VISA phenotype was confirmed by PAP-AUC. Results A total of 1005 isolates (39% MRSA) were studied. Susceptibility rates were: erythromycin 66.5%, clindamycin 79.2%, SXT 98.9%, rifampicin 97.3%, fusidic acid 67%, moxifloxacin 78.8%, vancomycin 99.9%, ceftaroline 92.9% and linezolid, tigecycline and daptomycin 100%. For mupirocin, high level resistance could be excluded for 98.9% of isolates. Vancomycin Etest® MIC50/90 were 1.5/1.5 mg/L, 58.5% of isolates exhibited a MIC > 1 and 8.7% a MIC of 2 mg/L, while Vitek® MIC50/90 were 1/1 and 3.1% showed MIC > 1 mg/L. One VISA strain was detected. Among the selected 175 isolates that were screened for hVISA phenotype, six (3.4%) were positive. In 315 bloodstream isolates, 64.1% had a vancomycin Etest® MIC > 1 mg/L. Conclusions This multi-centre surveillance study revealed that a significant percentage of contemporary S.aureus isolates from Greek patients have a vancomycin MIC (> 1 mg/L) that may compromise the clinical efficacy of the drug for the treatment of serious infections. The in vitro activity of SXT, rifampicin, mupirocin, linezolid, tigecycline, daptomycin and ceftaroline remains excellent.

Two hundred and fifty tracheal aspirates were subjected to direct antimicrobial susceptibility testing by disk diffusion, Etest and inoculation on antibiotic-enriched MacConkey agar plates. Results were compared with those obtained using an automated system on microorganisms recovered from standard quantitative culture. A total of 255 microorganisms were isolated from 194 positive samples by the standard quantitative procedure. A total of 85.1%, 82.5% and 72.5% agreement between direct disk diffusion, Etest and antibiotic-enriched MacConkey agar plates, respectively, and the standard procedure was observed in 64 microorganisms obtained from monomicrobial cultures that corresponded to 240 individual microorganism-antimicrobial agent combinations. Three (1.3%) and four (1.7%) very major errors for direct disk diffusion and Etest methods were observed, respectively. The antibiotic-enriched MacConkey agar plate method compared with the standard procedure demonstrated an unacceptable rate of very major (6.7%) and major errors (14.2%). Clinical evaluation of direct susceptibility tests based on the speculative impact on clinical practice by guiding patient's early treatment, if all positive cultures corresponded to infection, was correct in 79.9% for the direct disk diffusion test, 77.8% for the direct Etest method and 68.0% for antibiotic-enriched MacConkey agar plates. Direct diffusion tests (Etest or disk diffusion) applied on respiratory samples are rapid techniques that provide results comparable with standard antimicrobial susceptibility testing in

In this study, the performance of the chromogenic medium CHROMagar™ KPC was evaluated and was compared with in-house-daily prepared McConkey agar plates supplemented with imipenem (1 mg/L) for the detection of carbapenemase-producing Enterobacteriaceae. In this surveillance study, rectal swabs were cultured on both media and polymerase chain reaction (PCR) for bla(KPC) and bla(VIM) was used to confirm the genotype of growing colonies of Enterobacteriaceae. CHROMagar KPC was also tested with 17 genotypically characterised carbapenemase-producing and non-producing Gram-negative bacteria. It was shown that CHROMagar allows rapid detection of carbapenemase-producing Enterobacteriaceae, although bla(KPC)- and bla(VIM)-harbouring isolates could not be differentiated by colour or colony morphology. The positive and negative predictive values of the tested methods for the detection of carbapenemase-producing Enterobacteriaceae were, respectively, 100% and 98.8% for CHROMagar KPC and 94.7% and 88.6% for imipenem-supplemented McConkey agar. CHROMagar KPC medium is a useful screening medium for carbapenemase-producing Enterobacteriaceae in stools in settings with a high proportion of patients colonised with a variety of carbapenemase-producers.

BACKGROUND Although metallo-β-lactamases (MBLs) hydrolyse most β-lactams, including carbapenems, MBL-producing Enterobacteriaceae very often remain susceptible to carbapenems in vitro. We studied the in vivo efficacy of imipenem, meropenem, ertapenem and aztreonam against a carbapenem-susceptible MBL-producing clinical Escherichia coli strain in a rabbit intra-abdominal abscess model. METHODS Rabbits were inoculated intraperitoneally with 10(8) cfu/mL of VIM-1-positive E. coli and were assigned to receive no treatment (controls) or intravenous imipenem/cilastatin (imipenem) 70 mg/kg/12 h or meropenem 125 mg/kg/12 h or ertapenem 60 mg/kg/12 h or aztreonam 70 mg/kg/12 h. Dosing regimens were chosen on the basis of preliminary pharmacokinetic studies so that T(>MIC) was achieved for ≥50% of the dosing interval for all tested antibiotics. A total of eight doses were administered before sacrifice and the abscesses were harvested and quantitatively cultured. RESULTS MICs of imipenem, meropenem, ertapenem and aztreonam for the infecting isolate were 1, ≤0.25, 1.5 and ≤0.25 mg/L, respectively. The log(10) cfu/g (mean ± SD) viable counts in pus were as follows: controls (n = 16), 8.71 ± 1.34 (P

During a hospital-wide prospective point prevalence survey of faecal carriage and environmental colonisation of vancomycin-resistant enterococci in a tertiary care university hospital in Athens (Greece), five clinical and one environmental isolate from a light switch (all in the haematology ward) were identified as vancomycin- and linezolid-resistant vanA-positive Enterococcus faecium (VLRE). The studied isolates exhibited a linezolid minimum inhibitory concentration of 12microg/mL and carried at least one mutated copy of the 23S rRNA gene, as shown by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis to detect the G2576T mutation. The enterococcal surface protein (esp) gene was detected by PCR in all isolates. Molecular typing with pulsed field gel electrophoresis (PFGE) showed that the environmental and four of the five clinical isolates were genetically related. None of the colonised patients were previously exposed to linezolid, although heavy linezolid use was noted in the institution. A case-control study was performed to assess risk factors for VLRE colonisation. In univariate analysis, immunodeficiency, underlying haematological malignancy, duration of any antimicrobial treatment before VLRE isolation, and hospitalisation in the haematology ward were pointed out as possible risk factors. A multidisciplinary approach including intensified hand hygiene, patient contact isolation, disinfection of the inanimate environment and antibiotic restriction resulted in early containment of the VLRE colonisation outbreak.

The accuracy of disk susceptibility methods for colistin against 778 bacterial pathogens was evaluated in comparison with Etest using interpretive criteria available from the Clinical and Laboratory Standards Institute (CLSI). Colistin exhibited excellent activity against Acinetobacter baumannii and Escherichia coli isolates (minimum inhibitory concentration for 90% of the organisms (MIC(90))=0.5 mg/L), whilst it was less active both against Enterobacter spp. and Klebsiella pneumoniae (MIC for 50% of the organisms (MIC(50))=0.5 mg/L, MIC(90)=16 mg/L). Colistin also showed good activity against Pseudomonas aeruginosa (MIC(90)=2 mg/L, MIC(50)=1 mg/L) but poor activity against Stenotrophomonas maltophilia (MIC(50)=8 mg/L, MIC(90)=128 mg/L). Only 0.8% of minor errors were observed between the studied methods for P. aeruginosa isolates when the CLSI criteria were applied. All A. baumannii isolates with a zone diameteror =12 mm were resistant and those with a zone diameteror =14 mm were susceptible according to MIC breakpoints established by the CLSI. Among nine isolates exhibiting a zone diameter of 13 mm, one was resistant to colistin (MIC=8 mg/L) and eight isolates were susceptible (MIC=0.5 mg/L). Applying a MIC breakpoint ofor =2 mg/L for susceptibility in Enterobacteriaceae, all isolates with a zone diameteror =14 mm were susceptible, whilst all isolates with a zone diameteror =11 mm were resistant. Among isolates with zone diameters of 12-13 mm, 59% were characterised as susceptible. Major errors were observed only in K. pneumoniae isolates at a rate of 0.8%. The poor agar diffusion characteristics of colistin limit the predictive accuracy of the disk diffusion test and consequently values of 12-13 mm should be confirmed with MIC determination by Etest or broth dilution method.

Received 17 November 2007; returned 28 November 2007; revised 5 December 2007; accepted 11 December 2007 Objectives: Clinical isolates of Klebsiella pneumoniae (91), Escherichia coli (49), Enterobacter spp. (27), Proteus mirabilis (17), Citrobacter freundii (2), Providencia stuartii (3) and Serratia spp. (5), with various MICs of imipenem, were examined for production of metallo-b-lactamases (MBLs) with different phenotypic laboratory tests that have been previously published to detect MBLs in Pseudomonas aeruginosa and Acinetobacter spp. Methods: A total of 194 (95 MBL-positive and 99 MBL-negative) clinical isolates with imipenem MICs � 0.25 to >256 mg/L were examined. All isolates were evaluated by the double-disc synergy test (DDST), the combination disc test (CDT), the MBL Etest and the modified Hodge test. The presence of blaVIM and blaIMP genes was evaluated by in situ hybridization with specific probes and was certified by PCR. Results :I n 30blaVIM-positive isolates that exhibited MICs of imipenem � 4 mg/L, MBL Etest could not be evaluated. CDT with ceftazidime and 1900 or 750 mg of EDTA, and DDST after applying an imipenem disc 10 mm apart from a disc containing 1900 mg of EDTA, showed the highest sensitivity (97.9% to 100%) and specificity (87.9% to 96%) rates among the analysed procedures. CDT with imipenem and 1900 mg of EDTA exhibited a sensitivity of 94.7% and showed very good specificity (98%). Conclusions: The CDT with imipenem/imipenem10.5 M EDTA or ceftazidime/ceftazidime10.2 M EDTA and the DDST with imipenem 10 mm apart from EDTA are the most effective methods for the detection of MBLs in Enterobacteriaceae.

Vancomycin-resistant enterococci (VRE) have emerged as significant nosocomial pathogens. A hospital-wide prevalence study was performed to identify cases with VRE faecal colonisation. A case-control study using two randomly selected VRE-negative controls for each positive case was performed to assess risk-factors for VRE colonisation by univariate and multivariate analysis. VRE faecal colonisation was documented in 53 (14.3%) of 370 patients screened. Previous exposure to anti-anaerobic agents, as well as quinolones, was associated with VRE colonisation (p

Objectives: Infections due to multidrug-resistant (MDR) Gram-negative pathogens in the ICU have prompted the use of colistin, an antibiotic forgotten for decades. The aim of this retrospective observational study was to record and present the emergence of colistin-resistant Klebsiella pneumoniae (CRKB) in a Greek ICU. Methods: In a new university tertiary hospital, the first patients admitted in the ICU were already colonized or infected with MDR pathogens, and this led to frequent colistin use as part of empirical or microbiologically documented therapy. Colistin resistance was defined as MIC >4 mg/L by the Etest method. All CRKB isolated in surveillance cultures or clinical specimens in the ICU during the period 2004-5 were recorded along with patients' characteristics. Results: Eighteen CRKB were isolated from 13 patients over a 16 month period, representing either colonizing or infective isolates. Patients' mean age was 70 years, with a mean APACHE II score at admission of 22. They all had a long hospitalization (median 69 days) and a long administration of colistin (median 27 days). Colistin-resistant isolates were implicated as pathogens in two bacteraemias, a ventilator-associated pneumonia and two soft tissue infections. Repetitive extragenic palindromic PCR identified six distinct clones, and horizontal transmission was also documented. Conclusions: Selective pressure due to extensive or inadequate colistin use may lead to the emergence of colistin resistance among K. pneumoniae isolates, jeopardizing treatment options in the ICU, potentially increasing morbidity and mortality of critically ill patients and necessitating prudent use of colistin.

Nasopharyngeal Streptococcus pneumoniae isolates colonising young children are representative of isolates causing clinical disease. This study determined the frequency of macrolide-non-susceptible pneumococci, as well as their phenotypic and genotypic characteristics, among pneumococci collected during two cross-sectional surveillance studies of the nasopharynx of 2847 children attending day-care centres in the Athens metropolitan area during 2000 and 2003. In total, 227 macrolide-non-susceptible pneumococcal isolates were studied. Increases in macrolide non-susceptibility, from 23% to 30.3% (p

A clinical isolate of Enterobacter cloacae exhibiting reduced susceptibility to imipenem and a positive EDTA-disc synergy test was studied for carbapenemase production.MICs were determined with standard procedures as well as using a higher inoculum. Isoelectric focusing of cell extracts was used for detection of beta-lactamases. PCR assays with primers specific for the bla(VIM) gene and the conserved segments of class 1 integrons and sequence analyses were carried out to identify the gene and to map the metallo-beta-lactamase encoding integron. Transferability of the gene was assessed with conjugation experiments using the filter mating technique. To identify the location of the bla(VIM-1) gene, Southern hybridization was carried out in genomic DNA using an internal fragment of the bla(VIM-1) gene as a probe, amplified by PCR.The isolate was resistant to extended-spectrum beta-lactams. The MICs of carbapenems were below the resistance breakpoints but rose above resistance breakpoints when an inoculum of 10(8) cfu/mL was used. Isoelectric focusing detected a beta-lactamase with a pI of 6.1, which exhibited imipenem-hydrolysing activity in a microbiological assay. Ceftazidime and imipenem resistance were not transferable by conjugation. PCR assays identified the bla(VIM-1) gene in the variable region of a class 1 integron which also carried the aac(6')-IIc gene. The bla(VIM-1) probe hybridized with an approximately 130 kb fragment of genomic DNA, suggesting a chromosomal location of the gene.We describe a novel class 1 integron containing bla(VIM-1) and aac(6')-IIc genes in an E. cloacae clinical isolate.

To evaluate the impact of an antibiotic restriction policy on antibiotic consumption and Gram-negative resistance rates, in an environment of antibiotic overconsumption and increasing resistance rates for nosocomial pathogens.The study was a 'before and after' trial of 18-month duration; the antibiotic restriction policy program was implemented in 1998-2000 and was based on a government program addressed by the Ministry of Health to public hospitals on a national basis. This included prescribing of all newer antibiotics on an order form, auditing of the order forms and consultation with infectious diseases (ID) specialists, dispensing of treatment and prophylaxis guidelines, feedback, and face-to-face education. Antibiotic consumption and Gram-negative resistance rates were recorded before and after the intervention.Despite the addition of a new 40-bed ID department in the hospital during the 'after' period, the consumption of restricted antibiotics was significantly reduced by 42% (and their cost by 31%). Gram-negative resistance rates for Pseudomonas, Klebsiella, and Enterobacter, serving as index microorganisms for Gram-negative nosocomial pathogens, were significantly reduced during the 'after' period, even against antibiotics for which there was an increase in consumption.Multidisciplinary restriction programs can reduce antibiotic consumption and Gram-negative resistance rates in the hospital setting.

Here we describe a case of in vivo horizontal interspecies transmission of a KPC-2-producing plasmid from a Klebsiella pneumoniae to an Enterobacter aerogenes strain in the same patient. The patient's gut flora initially contained a carbapenem-susceptible E. aerogenes strain and 10 days after admission a KPC-2-positive K. pneumoniae. Three months after admission, a KPC-2-positive E. aerogenes was identified in fecal surveillance cultures. This isolate was isogenic with the initial E. aerogenes and contained a KPC-2-coding plasmid identical to that of the K. pneumoniae. The patient developed bacteraemia by the KPC-2-positive K. pneumoniae 17 days after her first colonization. In vivo horizontal transmission of blaKPC-carrying plasmids between bacterial species underscores the importance of antibiotic stewardship along with implementation of infection control measures for the containment of KPC-producers.

Background: Extended-spectrum b-lactamases (ESBLs) have emerged as an important mechanism of blactam resistance among community uropathogens. We characterized the ESBLs of a collection of Escherichia coli isolates recovered from outpatients with urinary tract infection during nationwide surveillance conducted from 2005 to 2006 in Greece, and evaluated the in vitro activity of mecillinam and mecillinam/clavulanate against them. Materials and methods: ESBLs were characterized with PCR and sequencing. In vitro interactions were evaluated with agar dilution with and without clavulanate (4 mg/L) using an inoculum of 10 4 or 10 6 cfu/spot as well as with time –kill methodology. Results: Among 48 ESBL producers, 47 (97.9%) were susceptible to mecillinam. CTX-M-type enzymes were produced by 87.2%, with CTX-M-3 being the most prevalent. SHV enzymes were found in 10.6%, VEB enzymes in 2.1%, TEM enzymes in 19.2% and OXA-type enzymes in 12.8%. Synergy with clavulanate was detected in 60.4% using the agar dilution method and in 43.8% using the time –kill methodology. An inoculum effect was detected in 64.6% of isolates, but this phenomenon was inverted and synergy was evidenced for 85.4% with clavulanate. When a high inoculum was used, 60.4% (29/48) were resistant to mecillinam, but 97.9% (47/48) were susceptible in the presence of clavulanate. Conclusions: CTX-M-type enzymes were the most prevalent among ESBL-producing E. coli uropathogens in Greece. Mecillinam may be useful in uncomplicated cystitis caused by ESBL producers with low MICs. The addition of the inhibitor could improve and extend the activity of mecillinam, even in the setting of infection with a high bacterial inoculum, and merits clinical evaluation.

16S ribosomal RNA methylase-mediated high-level resistance to 4-,6-aminoglycosides has been reported in clinical isolates of gram-negative bacilli from several countries. Three of 1534 (0.2%) isolates of Klebsiella pneumoniae and three of 734 (0.4%) Proteus mirabilis isolates from a university hospital in Athens, Greece, were positive for rmtB and highly resistant to all aminoglycosides tested (MICs ≥256 mg/L). Two of the K. pneumoniae rmtB-bearing isolates, were KPC-2 and OXA-10 producers and the third was a DHA-1 producer. One of the P. mirabilis isolates was a VIM-1 and OXA-10 producer and one was an OXA-10 producer. All rmtB-harbouring isolates were clonally unrelated. None of the E. coli (n = 1398) and Enterobacter spp. (n = 414) isolates were positive for armA, rmtA, rmtB, rmtC or rmtD.

The molecular epidemiology of VIM-producing Enterobacteriaceae isolated at the beginning of an epidemic in the Intensive Care Unit (ICU) of a university hospital in Athens, Greece, was studied. All Gram-negative organisms isolated from March 2004 to November 2005 positive for metallo-β-lactamase (MBL) production were submitted to polymerase chain reaction (PCR) and sequencing, to repetitive sequence-based PCR (Rep-PCR) for molecular typing, and to S1 nuclease digestion for plasmid DNA characterisation. Conjugation experiments and isoelectric focusing were performed to identify co-existing β-lactamases. Amongst 23 patients, 12 suffered one or more clinical infections. Eighty-two isolates representing one isolate per clone, source and ICU patient were studied, including Klebsiella pneumoniae (77), Enterobacter cloacae (2), Citrobacter freundii (1) and Pseudomonas aeruginosa (2). High clonal diversity was detected amongst the K. pneumoniae, with 10 distinct clones identified. Conjugation was successful in 54.5% of K. pneumoniae, and five different-sized plasmids were detected. All K. pneumoniae and both E. cloacae isolates shared the same bla(VIM-1)-containing class 1 integron structure also carrying aacA7, dhfrI and aadA1 gene cassettes. The C. freundii isolate carried a different integron that included bla(VIM-1) and aac(6')-IIc. Both P. aeruginosa isolates were positive for bla(VIM-2). It was not possible to identify specific clones with the potential to cause clinical infections. In conclusion, a multiclonal cluster of MBL-producers was responsible for the first cases of colonisation and/or infection in the ICU. A single integron structure, common in Greek hospitals, efficiently disseminated between clones and species, suggesting that the epidemic was mainly the result of successful horizontal transfer of mobile genetic material rather than the result of horizontal transfer of one or a few clones.

Background We describe the emergence and spread of Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing K. pneumoniae at a Greek University hospital. Methods Isolates with a carbapenem minimum inhibitory concentration >1 microg/mL and a negative EDTA-imipenem disk synergy test result were submitted to boronic acid disk test and to polymerase chain reaction (PCR) for KPC gene and sequencing. Records from patients who had KPC-2-producing K. pneumoniae isolated were retrospectively reviewed. Clinical isolates were submitted to molecular typing using pulsed-field gel electrophoresis, and the beta-lactamase content was studied using isoelectric focusing and PCR. Results From January 2007 through December 2008, 50 patients (34 in the intensive care unit [ICU]) were colonized (n = 32) or infected (n = 18) by KPC-2-producing K. pneumoniae. Increasing prevalence of KPC-2-producing K. pneumoniae coincided with decreasing prevalence of metallo-beta lactamase-producing isolates in our ICU. Multidrug resistance characterized the studied isolates, with colistin, gentamicin, and fosfomycin being the most active agents. Besides KPC-2, clinical isolates encoded TEM-1-like, SHV-11, SHV-12, CTX-M-15, and LEN-19 enzymes. Four different clonal types were detected; the predominant one comprised 41 single patient isolates (82%). Sporadic multiclonal cases of KPC-2-producing K. pneumoniae infection were identified from September 2007 through May 2008. The outbreak strain was introduced in February 2008 and disseminated rapidly by cross-transmission; 38 patients (76%) were identified after August 2008. Fourteen cases of bacteremia, 2 surgical site infections, 2 lower respiratory tract infections (1 bacteremic), and 1 urinary tract infection were identified. Most patients received a colistin-containing combination treatment. Crude mortality was 58.8% among ICU patients and 37.5% among non-ICU patients, but attributable mortality was 22.2% and 33.3%, respectively. Conclusions The emergence of KPC-2-producing K. pneumoniae in Greek hospitals creates an important challenge for clinicians and hospital epidemiologists, because it is added to the already high burden of antimicrobial resistance.

Although the rapid spread of carbapenemase-producing Gram-negatives (CPGNs) is providing the scientific community with a great deal of information about the molecular epidemiology of these enzymes and their genetic background, data on how to treat multidrug-resistant or extended drug-resistant carbapenemase-producing Enterobacteriaceae and how to contain their spread are still surprisingly limited, in spite of the rapidly increasing prevalence of these organisms and of their isolation from patients suffering from life-threatening infections. Limited clinical experience and several in vitro synergy studies seem to support the view that antibiotic combinations should be preferred to monotherapies. But, in light of the data available to date, it is currently impossible to quantify the real advantage of drug combinations in the treatment of these infections. Comprehensive clinical studies of the main therapeutic options, broken down by pathogen, enzyme and clinical syndrome, are definitely lacking and, as carbapenemases keep spreading, are urgently needed. This spread is unveiling the substantial unpreparedness of European public health structures to face this worrisome emergency, although experiences from different countries—chiefly Greece and Israel—have shown that CPGN transmission and cross-infection can cause a substantial threat to the healthcare system. This unpreparedness also affects the treatment of individual patients and infection control policies, with dramatic scarcities of both therapeutic options and infection control measures. Although correct implementation of such measures is presumably cumbersome and expensive, the huge clinical and public health problems related to CPGN transmission, alongside the current scarcity of therapeutic options, seem to fully justify this choice.

Background The dissemination of acquired metallo-beta-lactamases (MBLs) in members of the family Enterobacteriaceae is regarded as an emerging clinical threat. The clinical characteristics and outcomes of 17 cases of infection due to MBL-producing isolates were analyzed. Methods During a 3-year period, medical records for all patients with confirmed infection due to an MBL-producing strain belonging to the Enterobacteriaceae family were retrospectively analyzed. We screened for MBL production with the imipenem-ethylenediaminetetraacetic acid disk synergy test, and results were confirmed by polymerase chain reaction. Genetic relatedness between isolates was evaluated by repetitive extragenic palindromic polymerase chain reaction. Results Fourteen cases of bacteremia and 3 cases of ventilator-associated pneumonia due to an MBL-producing isolate were studied. Most of the patients had previously been colonized with an MBL-producing organism, and almost 60% had been exposed to carbapenems before infection. The isolated pathogens (Klebsiella pneumoniae, 14 cases; and Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes, 1 case each) exhibited variable minimum inhibitor concentrations of carbapenems (1 to >32 microg/mL) and resistance to most other beta-lactams. Tigecycline was active against all isolates, whereas colistin and gentamicin were active against 88% of them. Molecular studies confirmed the presence of a gene belonging to bla(VIM-1) cluster in all isolates. Among the 12 K. pneumoniae isolates, which were subjected to molecular typing, 11 distinct clones were identified. Five cases ( approximately 30%) occurred in patients who were already receiving carbapenem-containing treatment, and carbapenem treatment was considered to have failed. Twelve cases were treated with a colistin-containing regimen. The attributable mortality rate was 18.8%. Conclusions MBL-producing Enterobacteriaceae can cause severe, often fatal infection in severely ill patients, irrespective of the MIC of carbapenems. Colonization with an MBL-producer is a preceding event, highlighting the importance of surveillance. Both infection control practices and antibiotic policies should be intensified to contain the spread of these problematic bacteria.

carbapenemases, Enterobacteriaceae*Correspondence address. Fourth Department of InternalMedicine, University General Hospital ‘ATTIKON’, Rimini 1,124 62 Chaidari, Greece. Tel: þ32105326426; Fax:þ32105326446; E-mail: hgiama@ath.forthnet.grSir,Until now, there have been only a few reports on the productionof VIM-type metallo-b-lactamases (MBLs) in members of thefamily Enterobacteriaceae.

Lamprini Galani , Irene Galani , Maria Souli , Ilias Karaiskos , Emanouella Katsouda , Eleni Patrozou , Fotini Baziaka , Charalampos Paskalis , Helen Giamarellou * a 6th Department of Internal Medicine, Hygeia Hospital, 4 Erythrou Stavrou Street & Kifisias Avenue, 151 23 Marousi, Athens, Greece b Intensive Care Unit, Hygeia Hospital, 4 Erythrou Stavrou Street & Kifisias Avenue, 151 23 Marousi, Athens, Greece Hygeia Hospital, 4 Erythrou Stavrou Street & Kifisias Avenue, 151 23 Marousi, Athens, Greece