In vitro activity of apramycin (EBL-1003) in combination with colistin, meropenem, minocycline or sulbactam against XDR/PDR Acinetobacter baumannii isolates from Greece.

OBJECTIVES: To evaluate the in vitro activity of the combination of apramycin with colistin, meropenem, minocycline or sulbactam, against some well-characterized XDR Acinetobacter baumannii clinical isolates from Greece, to understand how apramycin can be best incorporated into clinical practice and optimize effectiveness. METHODS: In vitro interactions of apramycin (0.5×, 1× and 2× the MIC value) with colistin (2 mg/L), meropenem (30 mg/L), minocycline (3.5 mg/L) or sulbactam (24 mg/L) were tested using time-kill methodology. Twenty-one clinical A. baumannii isolates were chosen, exhibiting apramycin MICs of 4-16 mg/L, which were at or below the apramycin preliminary epidemiological cut-off value of 16 mg/L. These isolates were selected for a range of colistin (4-32 mg/L), meropenem (16-256 mg/L), minocycline (8-32 mg/L) and sulbactam (8-32 mg/L) MICs across the resistant range. Synergy was defined as a ≥2 log10 cfu/mL reduction compared with the most active agent. RESULTS: The combination of apramycin with colistin, meropenem, minocycline or sulbactam was synergistic, at least at one of the concentrations of apramycin (0.5×, 1× or 2× MIC), against 83.3%, 90.5%, 90.9% or 92.3% of the tested isolates, respectively. Apramycin alone was bactericidal at 24 h against 9.5% and 33.3% of the tested isolates at concentrations equal to 1× and 2× MIC, while the combination of apramycin at 2× MIC with colistin, meropenem or sulbactam was bactericidal against all isolates tested (100%). The apramycin 2× MIC/minocycline combination had bactericidal activity against 90.9% of the tested isolates. CONCLUSIONS: Apramycin combinations may have potential as a treatment option for XDR/pandrug-resistant (PDR) A. baumannii infections and warrant validation in the clinical setting, when this new aminoglycoside is available for clinical use.

Ceftazidime/avibactam in the era of carbapenemase-producing Klebsiella pneumoniae: experience from a national registry study.

BACKGROUND: Infections caused by KPC-producing Klebsiella pneumoniae (Kp) are associated with high mortality. Therefore, new treatment options are urgently required. OBJECTIVES: To assess the outcomes and predictors of mortality in patients with KPC- or OXA-48-Kp infections treated with ceftazidime/avibactam with an emphasis on KPC-Kp bloodstream infections (BSIs). METHODS: A multicentre prospective observational study was conducted between January 2018 and March 2019. Patients with KPC- or OXA-48-Kp infections treated with ceftazidime/avibactam were included in the analysis. The subgroup of patients with KPC-Kp BSIs treated with ceftazidime/avibactam was matched by propensity score with a cohort of patients whose KPC-Kp BSIs had been treated with agents other than ceftazidime/avibactam with in vitro activity. RESULTS: One hundred and forty-seven patients were identified; 140 were infected with KPC producers and 7 with OXA-48 producers. For targeted therapy, 68 (46.3%) patients received monotherapy with ceftazidime/avibactam and 79 (53.7%) patients received ceftazidime/avibactam in combination with at least another active agent. The 14 and 28 day mortality rates were 9% and 20%, respectively. The 28 day mortality among the 71 patients with KPC-Kp BSIs treated with ceftazidime/avibactam was significantly lower than that observed in the 71 matched patients, whose KPC-Kp BSIs had been treated with agents other than ceftazidime/avibactam (18.3% versus 40.8%; P = 0.005). In the Cox proportional hazards model, ultimately fatal disease, rapidly fatal disease and Charlson comorbidity index ≥2 were independent predictors of death, whereas treatment with ceftazidime/avibactam-containing regimens was the only independent predictor of survival. CONCLUSIONS: Ceftazidime/avibactam appears to be an effective treatment against serious infections caused by KPC-Kp.

Assessment of follicle viability using fluorescence microscopy before and after ovarian thawing.

The incidence of young women diagnosed with cancer has been globally increasing. In many cases the surgical approach followed by chemotherapy, radiotherapy or hormonal therapy could lead to infertility or premature ovarian failure. Several options are available in order to preserve fertility and increase the future gestation rate. Among embryo cryopreservation and oocyte cryopreservation, ovarian tissue cryopreservation represents an ideal option, especially for premenopausal women and for those who cannot delay the start of chemotherapy. The purpose of this study was to examine the follicle viability using fluorescence microscope before and after ovarian thawing.

Time-kill effect of levofloxacin on multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii: synergism with imipenem and colistin.

In the present study, we challenged the concept that levofloxacin should not be used for the management of ventilator-associated pneumonia (VAP) when minimum inhibitory concentrations (MICs) exceed 2 µg/ml. Multidrug-resistant (MDR) and genetically distinct isolates of Pseudomonas aeruginosa (n = 49) and Acinetobacter baumannii (n = 29) from patients with VAP were exposed over time to levofloxacin, imipenem, colistin and their combinations. Synergy between levofloxacin and imipenem was found in 55.3 % and between levofloxacin and colistin in 90.9 % of isolates of P. aeruginosa within the first 4 h of growth. Synergy with imipenem but not with colistin was dependent of the MIC. Synergy between levofloxacin and imipenem was found in 58.6 % of isolates of A. baumannii after 24 h of growth. Considerable synergy was found between levofloxacin and colistin, reaching 84.8 % of isolates of A.baumannii after 6 h of growth. Synergy was independent from the MIC. These results create hopes that levofloxacin can be used as combination therapy for infections by MDR bacteria.

Genomic characterization of a KPC-23-producing Klebsiella pneumoniae ST258 clinical isolate resistant to ceftazidime-avibactam.

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.

Risk-factors and predictors of mortality in patients colonised with vancomycin-resistant enterococci.

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 <0.05). The presence of an invasive device (OR 4.8, p 0.003) and the duration of any antimicrobial treatment before VRE isolation (OR 1.2, p <0.001) predicted VRE colonisation in multivariate models. The crude mortality rate for patients with VRE colonisation was 24.5%, but VRE colonisation was not an independent predictor of mortality in these patients. These results suggest that an active surveillance programme focusing on specific patient groups may help in the identification of VRE-colonised patients. Promptly implemented infection control strategies targeting these groups should help to combat the rising incidence of VRE.

Emergence of extensively drug-resistant and pandrug-resistant Gram-negative bacilli in Europe.

International and local surveillance networks as well as numerous reports in the biomedical literature provide evidence that the prevalence of antibiotic resistant Gram-negative bacteria is escalating in many European countries. Furthermore, isolates characterised as multidrug-resistant (i.e. resistant to three or more classes of antimicrobials), extensively drug resistant (i.e. resistant to all but one or two classes) or pandrug-resistant (i.e. resistant to all available classes) are increasingly frequently isolated in hospitalised patients causing infections for which no adequate therapeutic options exist. Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae are specifically addressed in this review as the most problematic and often extensively or pandrug-resistant pathogens. According to the available multicentre surveillance studies, the proportion of imipenem-resistant A. baumannii strains is reported to be as high as 85% in bloodstream isolates from intensive care unit patients in Greece and 48% in clinical isolates from hospitalised patients in Spain and Turkey. Among 33 European countries participating in the European Antimicrobial Resistance Surveillance System (EARSS) in 2007, six countries reported carbapenem resistance rates of more than 25% among P. aeruginosa isolates, the highest rate reported from Greece (51%). According to EARSS, Greece has also the highest resistance rates among K. pneumoniae; 46% to carbapenems, 58% to quinolones and 63% to third generation cephalosporins. This review describes the magnitude of antimicrobial resistance in Gram-negative bacteria in Europe highlighting where the efforts of the scientific communities, the academia, the industry and the government should focus in order to confront this threat.

Characterisation of macrolide-non-susceptible Streptococcus pneumoniae colonising children attending day-care centres in Athens, Greece during 2000 and 2003.

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 <0.05), and in macrolide and penicillin co-resistance, from 3.4% to 48.6% (p <0.001), were identified during the study period. The M resistance phenotype, associated with the presence of the mef(A)/(E) gene, predominated in both surveys, and isolates carrying both mef(A)/(E) and erm(AM) were identified, for the first time in Greece, among the isolates from the 2003 survey. Pulsed-field gel electrophoresis analysis of the isolates from the 2000 survey indicated the spread of a macrolide- and penicillin-resistant clone among day-care centres. The serogroups identified most commonly in the study were 19F, 6A, 6B, 14 and 23F, suggesting that the theoretical protection of the seven-valent conjugate vaccine against macrolide-non-susceptible isolates was c. 85% during both study periods.

First identification of an Escherichia coli clinical isolate producing both metallo-beta-lactamase VIM-2 and extended-spectrum beta-lactamase IBC-1.

An Escherichia coli strain with decreased susceptibility to carbapenems was isolated from a hospitalised patient in Athens, Greece. The strain was resistant to all beta-lactams, including aztreonam, whereas the MIC of imipenem and meropenem was 0.5 mg/L. A positive EDTA-disk synergy test suggested the production of a metallo-beta-lactamase. PCR experiments revealed the presence of the bla(VIM-2), bla(IBC-1), and bla(TEM-1) genes. Resistance to beta-lactams was not transferable by conjugation. This is the first report of a clinical isolate of E. coli producing VIM-2, and the first report of the coexistence of bla(VIM-2) and bla(IBC-1) in a single clinical isolate.

Transferable plasmid mediating resistance to multiple antimicrobial agents in Klebsiella pneumoniae isolates in Greece.

OBJECTIVE: To investigate the underlying resistance mechanisms in 10 Klebsiella pneumoniae isolates. METHODS: Ten K. pneumoniae strains according to distinct bacteriocin typing and REP-PCR, were examined for their plasmid content, their ability to transfer their resistance to aminoglycosides and third-generation cephalosporins, and their production of aminoglycoside-modifying enzymes and beta-lactamases. RESULTS: Transfer of resistance to the above-mentioned antibiotics as well as to co-trimoxazole and tetracycline in Escherichia coli strain RC 85 at a frequency of 5-106 was achieved for all strains by conjugation. Similar strains harbor a self-transferable multiresistant plasmid (80 kb) with similar EcoRI and HindIII restriction patterns. This plasmid encodes an extended-spectrum beta-lactamase which confers high-level resistance to third-generation cephalosporins and aztreonam. It produces SHV-5 beta-lactamase, as demonstrated by isoelectric focusing and DNA sequencing. Aminoglycoside resistance was co-transferred, and AAC(6')-I, mediating resistance to gentamicin, tobramycin, netilmicin and amikacin, and AAC(3)-I, mediating resistance to gentamicin and sisomycin, were encoded in all isolates and their transconjugants, while APH(3')-I, mediating resistance to kanamycin and neomycin, was encoded in seven strains. CONCLUSIONS: It appears that a multiresistant transferable plasmid encoding the SHV-5 beta-lactamase, causing unusually high resistance to ceftazidime and aztreonam, and the combination AAC(6')-I + AAC(3)-I of acetylating enzymes causing, also resistance to all clinically available aminoglycosides, is established in K. pneumoniae in Greece.

In vitro interaction of colistin and rifampin on multidrug-resistant Pseudomonas aeruginosa.

The administration of colistin is considered as the last alternative for infections by multidrug-resistant isolates of Pseudomonas aeruginosa; however its use is limited due to its considerable toxicity and poor pharmacokinetics. In order to define the in vitro activity of colistin combined with rifampin, 28 isolates resistant to piperacillin, ceftazidime, imipenem, meropenem, ciprofloxacin, amikacin, rifampin and colistin were tested. Seventeen of them that were found by PFGE to be genetically distinct were over time exposed to 2 microg/ml of colistin, to 2 microg/ml of rifampin and to their combination. Applied concentrations were selected to correspond to the mean serum level of the tested antimicrobials. Synergy between colistin and rifampin was found in four (23.5%), six (35.3%), seven (41.7%) and two (11.8%) isolates after 2, 4, 6 and 24 hours of growth, respectively. Bacterial re-growth was detected after 24 hours of exposure to the tested interaction. It is concluded that colistin and rifampin express a considerable in vitro synergistic effect on multidrug-resistant P. aeruginosa. The reported interaction should be tested in animal studies before introduction in clinical practice.

Prevalence of 16S rRNA methylase genes in Enterobacteriaceae isolates from a Greek university hospital.

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.

Colonization and infection by colistin-resistant Gram-negative bacteria in a cohort of critically ill patients.

In recent years there has been renewed interest in colistin for the treatment of infections by multidrug-resistant Gram-negative bacteria, causing concern that increasing use may be accompanied by the emergence of resistance. This is a retrospective cohort study of colonization and infection by colistin-resistant (CR) gram-negative bacteria in critically ill patients. Colonization data were based on surveillance culture results. Among 150 patients, 78 (52%) were colonized by CR Gram-negative bacteria. Among them, 30 (20%) were colonized by Klebsiella pneumoniae isolates and 51 (34%) were colonized by intrinsically resistant to colistin (CIR) enterobacteriaceae. Seven cases of infection were caused by CR K. pneumoniae and 12 cases by CIR strains. The main risk factor for colonization by CR pathogens was colistin treatment.

Presence of plasmid-mediated quinolone resistance in Klebsiella pneumoniae and Escherichia coli isolates possessing blaVIM-1 in Greece.

Amongst nalidixic acid-resistant, ciprofloxacin-susceptible Escherichia coli and Klebsiella pneumoniae clinical isolates recovered over a 5-month period from inpatients and outpatients of Attikon University General Hospital (Athens, Greece), only one E. coli was positive for qnrB2 and one K. pneumoniae was positive for qnrA1. Both isolates were extended-spectrum beta-lactamase-negative, metallo-beta-lactamase-positive and carried the bla(VIM-1) gene. Neither of the isolates had mutations in gyrA and parC or carried aac(6')-Ib-cr or qepA. The K. pneumoniae isolate also harboured bla(CMY-13) on the same transferable plasmid with qnrA1. This is the first report of a qnrA1-positive K. pneumoniae and qnrB2-positive E. coli harbouring a concurrent bla(VIM-1) gene.

Acquired carbapenemases in Gram-negative bacterial pathogens: detection and surveillance issues.

Acquired carbapenemases are emerging resistance determinants in Gram-negative pathogens, including Enterobacteriaceae, Pseudomonas aeruginosa and other Gram-negative non-fermenters. A consistent number of acquired carbapenemases have been identified during the past few years, belonging to either molecular class B (metallo-beta-lactamases) or molecular classes A and D (serine carbapenemases), and genes encoding these enzymes are associated with mobile genetic elements that allow their rapid dissemination in the clinical setting. Therefore, detection and surveillance of carbapenemase-producing organisms have become matters of major importance for the selection of appropriate therapeutic schemes and the implementation of infection control measures. As carbapenemase production cannot be simply inferred from the resistance profile, criteria must be established for which isolates should be suspected and screened for carbapenemase production, and for which tests (phenotypic and/or genotypic) should be adopted for confirmation of the resistance mechanism. Moreover, strategies should be devised for surveillance of carbapenemase producers in order to enable the implementation of effective surveillance programmes. The above issues are addressed in this article, as a follow-up to an expert meeting on acquired carbapenemases that was recently organized by the ESCMID Study Group for Antibiotic Resistance Surveillance.

Transposon mutagenesis and strain construction in Zymomonas mobilis.

Conjugative or mobilizable plasmids carrying the transposable elements Tn5, Tn501 or mini Mu were readily transferred from Escherichia coli donors into Zymomonas mobilis recipients with frequencies depending both on donor and recipient strain used. With the exception of pULB113 (RP4::mini Mu), all foreign plasmids exhibited high instability in Z. mobilis transconjugants under both selective and non-selective conditions. Transposition events and consequent mutagenesis occurred readily in Z. mobilis transconjugant strains, with Tn5 and Tn501 being far less successful than mini Mu. Transposon mutagenesis with the help of mini Mu resulted in the isolation of a large number of independent auxotrophs with polyauxotrophs, cysteine, methionine and isoleucine requiring-isolates being the most frequent. When chromosomal DNA from all these mutants was digested with various restriction enzymes and the resulting restriction patterns were hybridized with a mini Mu probe, the majority of these mutants appeared to have insertions at different sites of the chromosome. Thus, transposon mutagenesis by mini Mu is proven to be a simple and efficient tool for mutant production and the genetic analysis of Z. mobilis.

Stimulation of innate immunity by susceptible and multidrug-resistant Pseudomonas aeruginosa: an in vitro and in vivo study.

In attempt to investigate the stimulatory effect of Pseudomonas aeruginosa on innate immunity and to correlate it to its level of resistance to antimicrobials, 20 isolates were applied; 8 isolates were susceptible and 12 multidrug-resistant. Genetic diversity was defined by PFGE. Human monocytes of two healthy volunteers were in vitro stimulated by the isolates for the production of pro-inflammatory (TNF-alpha, IL-1beta, IL-6, IL-8 and IL-12) and anti-inflammatory cytokines (IL-10), of malondialdehyde and of procalcitonin. Cytokines were estimated by EIA, malondialdehyde by the thiobarbiturate assay and procalcitonin by an immunochemiluminometric assay. Survival of 48 Wistar rats was recorded after induction of sepsis by the intraperitoneal injection of three susceptible and three multidrug-resistant isolates. To test whether comparative effect of the latter isolates on survival correlates with any difference of monocyte-mediated release of pro-inflammatory mediators, monocytes of two rats were in vitro stimulated for the production of TNF-alpha and of malondialdehyde. In vitro stimulation of human monocytes by the susceptible isolates elicited elevated production of malondiadeheyde, of IL-1beta and of IL-6 compared to stimulation by multidrug-resistant isolates. Similar differences were found for TNF-alpha and IL-8, but they were not statistically significant. Production of IL-10 and IL-12 was not detected after stimulation with any isolate. Levels of procalcitonin were similar after induction with either susceptible or multidrug-resistant isolates. Mean survival of animals was 7.56, 21.80 and 55.20 h, respectively, after challenge by the susceptible isolates and 28.89, 61.8 and more than 120 h, respectively, after challenge by the multidrug-resistant isolates. Differences of survival were accompanied by greater rodent monocyte-release of TNF-alpha and malondialdehyde after stimulation by the susceptible isolates compared to multidrug-resistant ones. It is concluded that considerable differences are encountered on the stimulation of human monocytes by susceptible and resistant isolates of Pseudomonas aeruginosa. These results correlate with in vivo evidence and might influence decision on therapeutics.