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Centre for Genomic Pathogen Surveillance and the Euro-GASP study group: Sonja Pleininger, Alexander Indra, Irith De Baetselier, Wim Vanden Berghe, Blaženka Hunjak, Tatjana Nemeth Blažić, Panayiota Maikanti-Charalambous, Despo Pieridou, Hana Zákoucká, Helena Žemličková, Steen Hoffmann, Susan Cowan, Lasse Jessen Schwartz, Rita Peetso, Jevgenia Epstein, Jelena Viktorova, Ndeindo Ndeikoundam, Beatrice Bercot, Cécile Bébéar, Florence Lot, Susanne Buder, Klaus Jansen, Vivi Miriagou, Georgios Rigakos, Vasilios Raftopoulos, Eszter Balla, Mária Dudás, Lena Rós Ásmundsdóttir, Guðrún Sigmundsdóttir, Guðrún Svanborg Hauksdóttir, Thorolfur Gudnason, Aoife Colgan, Brendan Crowley, Sinéad Saab, Paola Stefanelli, Anna Carannante, Patrizia Parodi, Gatis Pakarna, Raina Nikiforova, Antra Bormane, Elina Dimina, Monique Perrin, Tamir Abdelrahman, Joël Mossong, Jean-Claude Schmit, Friedrich Mühlschlegel, Christopher Barbara, Francesca Mifsud, Alje Van Dam, Birgit Van Benthem, Maartje Visser, Ineke Linde, Hilde Kløvstad, Dominique Caugant, Beata Młynarczyk-Bonikowska, Jacinta Azevedo, Maria-José Borrego, Marina Lurdes Ramos Nascimento, Peter Pavlik, Irena Klavs, Andreja Murnik, Samo Jeverica, Tanja Kustec, Julio Vázquez Moreno, Asuncion Diaz, Raquel Abad, Inga Velicko, Magnus Unemo, Helen Fifer, Jill Shepherd, Lynsey Patterson Background: Genomic surveillance using quality-assured whole-genome sequencing (WGS) together with epidemiological and antimicrobial resistance (AMR) data is essential to characterise the circulating Neisseria gonorrhoeae lineages and their association to patient groups (defined by demographic and epidemiological factors). In 2013, the European gonococcal population was characterised genomically for the first time. We describe the European gonococcal population in 2018 and identify emerging or vanishing lineages associated with AMR and epidemiological characteristics of patients, to elucidate recent changes in AMR and gonorrhoea epidemiology in Europe. Methods: We did WGS on 2375 gonococcal isolates from 2018 (mainly Sept 1-Nov 30) in 26 EU and EEA countries. Molecular typing and AMR determinants were extracted from quality-checked genomic data. Association analyses identified links between genomic lineages, AMR, and epidemiological data. Findings: Azithromycin-resistant N gonorrhoeae (8·0% [191/2375] in 2018) is rising in Europe due to the introduction or emergence and subsequent expansion of a novel N gonorrhoeae multi-antigen sequence typing (NG-MAST) genogroup, G12302 (132 [5·6%] of 2375; N gonorrhoeae sequence typing for antimicrobial resistance [NG-STAR] clonal complex [CC]168/63), carrying a mosaic mtrR promoter and mtrD sequence and found in 24 countries in 2018. CC63 was associated with pharyngeal infections in men who have sex with men. Susceptibility to ceftriaxone and cefixime is increasing, as the resistance-associated lineage, NG-MAST G1407 (51 [2·1%] of 2375), is progressively vanishing since 2009-10. Interpretation: Enhanced gonococcal AMR surveillance is imperative worldwide. WGS, linked to epidemiological and AMR data, is essential to elucidate the dynamics in gonorrhoea epidemiology and gonococcal populations as well as to predict AMR. When feasible, WGS should supplement the national and international AMR surveillance programmes to elucidate AMR changes over time. In the EU and EEA, increasing low-level azithromycin resistance could threaten the recommended ceftriaxone-azithromycin dual therapy, and an evidence-based clinical azithromycin resistance breakpoint is needed. Nevertheless, increasing ceftriaxone susceptibility, declining cefixime resistance, and absence of known resistance mutations for new treatments (zoliflodacin, gepotidacin) are promising. This study was supported by the European Centre for Disease Prevention and Control, the Centre for Genomic Pathogen Surveillance, the Li Ka Shing Foundation (Big Data Institute, University of Oxford), the Wellcome Genome Campus, the Foundation for Medical Research at Örebro University Hospital, and grants from Wellcome (098051 and 099202). LSB was funded by Conselleria de Sanitat Universal i Salut Pública, Generalitat Valenciana (Plan GenT CDEI-06/20-B), Valencia, Spain, and Ministry of Science, Innovation and Universities (PID2020–120113RA-I00), Spain, at the time of analysing and writing this manuscript. info:eu-repo/semantics/publishedVersion

We survey the diversity of antimicrobial resistance profiles of Escherichia coli ( E. coli) isolates and molecular genotypes by means of phylogenetic groups circulating in the environment, in sewage and receiving waters bodies as well as clinical isolates both from Boeotia regional district of Greece. The predominant resistant profile among all environmental isolates was to penicillins - ampicillin (AMP) and piperacillin (PIP). ESBLs resistance related profiles were observed both in environmental and clinical isolates. Phylogenetic group B2 was the predominant in clinical isolates, whilst group A was dominant in environmental isolates. We report that reclaimed river water and wastewater are reservoirs of R and MDR E. coli to commonly used antibiotics in clinical practice posing putative threats for human and animal health.

In the absence of a molecule that would collectively inhibit both metallo-β-lactamases and serine reactive carbapenemases, containment of their genes’ spreading is the main weapon currently available for confronting carbepenem resistance in hospitals. Cost effective methodologies rapidly detecting carbapenemase producing enterobacteria (CPE) would facilitate such measures. Herein a low cost CPE detection method was developed that was based on the direct colorimetry of the yellow shift caused by the accumulation of diketopiperazines – products of the acid catalyzed imipenem oligomerization – induced by carbapenemase action on dense solutions of imipenem/cilastatin. The reactions were studied by spectrophotometry in the visible spectrum using preparations of β-lactamases from the four molecular classes. The effects of various buffers on reactions containing the potent carbapenemases NDM-1 and NMC-A were monitored at 405 nm. Optimal conditions were used for the analysis of cell suspensions and the assay was evaluated using 38 selected enterobacteria including 29 CPE as well as nine carbapenemase-negative strains overexpressing other β-lactamases. The development of the yellow color was specific for carbapenemase containing enzyme preparations and the maximum intensity was achieved in acidic or un-buffered conditions in the presence of zinc. When applied on bacterial cell suspensions the assay could detect CPE with 96.7 % sensitivity and 100 % specificity with results being comparable to those obtained with the CARBA NP technique. Direct colorimetry of carbapenemase-induced imipenem decomposition required minimum reagents while exhibited high accuracy in detecting CPE. Therefore it should be considered for screening purposes after further clinical evaluation.ImportanceCurrently, spread of multi-drug resistant (MDR) carbapenemase-producing enterobacteria (CPE), mostly in the clinical setting, is among the most pressing public health problems worldwide. In order to effectively control CPE, use of reliable and affordable methods detecting carbapenemase genes or the respective β-lactamases is of vital importance. Herein we developed a novel method, based on a previously undescribed phenomenon, which can detect CPE with few reagents by direct colorimetry of bacterial suspensions and imipenem/cilastatin mixtures.

The timely and accurate detection of carbapenemase-producing Enterobacterales (CPE) is imperative to manage this worldwide problem in an effective fashion. Herein we addressed the question of whether the protons produced during imipenem hydrolysis could be detected using an ion sensitive field effect transistor (ISFET). Application of the methodology on enzyme preparations showed that the sensor is able to detect carbapenemases of the NDM, IMP, KPC and NMC-A types at low nanomolar concentrations while VIM and OXA-48 responded at levels above 100 nM. Similar results were obtained when CPE cell suspensions were tested; NDM, IMP, NMC-A and KPC producers caused fast reductions of the output potential. Reduction rates with VIM-type and especially OXA-48 producing strains were significantly lower. Based on results with selected CPEs and carbapenemase-negative enterobacteria, a threshold of 10 mV drop at 30 min was set. Applying this threshold, the method exhibited 100% sensitivity for NDM, IMP and KPC and 77.3% for VIM producers. The OXA-48-positive strains failed to pass the detection threshold. A wide variety of carbapenemase-negative control strains were all classified as negative (100% specificity). In conclusion, an ISFET-based approach may have the potential to be routinely used for non OXA-48-like CPE detection in the clinical laboratory.

Pathogens able to cross the blood-brain barrier (BBB) induce long-term neurological sequelae and death. Understanding how neurotropic pathogens bypass this strong physiological barrier is a prerequisite to devise therapeutic strategies. Here we propose an innovative model of infection in the developing Drosophila brain, combining whole brain explants with in vivo systemic infection. We find that several mammalian pathogens are able to cross the Drosophila BBB, including Group B Streptococcus (GBS). Amongst GBS surface components, lipoproteins, and in particular the B leucine-rich Blr, are important for BBB crossing and virulence in Drosophila. Further, we identify (V)LDL receptor LpR2, expressed in the BBB, as a host receptor for Blr, allowing GBS translocation through endocytosis. Finally, we show that Blr is required for BBB crossing and pathogenicity in a murine model of infection. Our results demonstrate the potential of Drosophila for studying BBB crossing by pathogens and identify a new mechanism by which pathogens exploit the machinery of host barriers to generate brain infection., Bacterial and fungal pathogens that cross the blood-brain-barrier (BBB) can cause severe disease. Here, Benmimoun et al. develop a model to study BBB crossing in the developing Drosophila brain and discover Group B Streptococcus factors important for BBB crossing and virulence, one of which, a lipoprotein, they confirm in mice.

Pathogens able to cross the blood-brain barrier (BBB) induce long-term neurological sequelae and death. Understanding how neurotropic pathogens bypass this strong physiological barrier is a prerequisite to devise therapeutic strategies. Here we propose an innovative model of infection in the developing Drosophila brain, combining whole brain explants within vivosystemic infection. We identified several mammalian pathogens able to cross the Drosophila BBB, including Group B Streptococcus (GBS). Amongst GBS surface components, lipoproteins, and in particular the B leucin-rich Blr, were important for BBB crossing and virulence in Drosophila. Further, we identified (V)LDL receptor LpR2, expressed in the BBB, as a host receptor for Blr, allowing GBS translocation through endocytosis. Finally, we demonstrated that Blr is required for BBB crossing and pathogenicity in a murine model of infection. Our results support the relevance of Drosophila for studying host-pathogen interactions and identify a new mechanism by which pathogens exploit host barriers to generate brain infection.

Background Carbapenem-resistant Acinetobacter baumannii (CRAB) has gained global notoriety as a critically important nosocomial pathogen. It mostly affects debilitated patients, causing pneumonia and bloodstream infections with high mortality rates. Difficulties in treating CRAB infections stem from a formidable resistance profile that leaves available only a few antibiotics of uncertain efficacy such as colistin and tigecycline. Despite the relentless attempts to improve therapeutic approaches (as depicted in colistin-oriented randomized clinical trials and the numerous observational studies), progress is still limited. Aims We aim (a) to assist physicians to adapt therapeutic approaches in CRAB infections by considering all potentially available antimicrobials, and (b) to present directions for future investigations that emerge through treatment efforts in endemic settings. Sources Articles and reviews from PubMed and Scopus databases; studies from ClinicalTrials.gov; presentations from ECCMID congresses and IDWeek meetings. Content The review provides a succinct overview of the important pharmacokinetic/pharmocodynamic parameters of relevant antimicrobial agents, a critical appraisal of randomized control trials and observational studies, suggestions for increasing the strength of observational studies and directions facilitating the choice of therapeutic regimens by severity of infection and status of the host. Implications The lack of an optimal therapeutic regimen for CRAB thus far, as shown in this review, suggests the need to thoroughly investigate alternative approaches through carefully designed trials that should include all relevant drugs. Some of these alternative directions are indicated in the present review.

Diabetic foot ulceration is a common and severe complication of diabetes, causing substantial social, medical, and economic burdens. Treatment of foot ulcers remains challenging, thus requiring increasing awareness and more efficient management. This study investigates the efficacy of ointments, containing as main active ingredient the olive oil extract of the marine isopod Ceratothoa oestroides, in the treatment of patients with diabetic foot ulcers. Fifty-two patients were allocated into four treatment groups either receiving therapy with an ointment containing extract of C. oestroides or extract of C. oestroides and eosin or extract of C. oestroides and cefaclor or no treatment. Patients were monitored for a period of 135 days by evaluation of transepidermal water loss, skin hydration, planimetry, photo-documentation, and clinical condition. Treatment with the extract of C. oestroides demonstrated significant healing properties that became evident after 45 days of treatment and resulted in complete ulcer healing in 61% of the patients. A significant improvement in transepidermal water loss (p < 0.001), skin hydration levels (p < 0.001), and wound area (p < 0.001) was observed in all patients. Similar efficacy was demonstrated for the combination of C. oestroides extract with eosin treatment (p < 0.001). On the contrary, the combination of C. oestroides extract with cefaclor antibiotic agent completely inhibited the healing properties of the isopod extract and did not improve water loss, skin hydration, or wound area. An important factor for C. oestroides extract healing properties is its selective activity against Gram negative bacteria. Ointments containing C. oestroides extract alone or combined with the antimicrobial agent eosin emerges as an effective regimen for the treatment of diabetic foot ulcers.

The cephalosporinase CMY-107, a Tyr199Cys mutant form of CMY-2 encoded by an IncI self-transferable plasmid carried by an Escherichia coli clinical strain, was characterized. The enzyme hydrolyzed oximino-cephalosporins and aztreonam more efficiently than CMY-2 did.

In an effort to identify the optimal cyclodextrin (CD) host for delivery of penicillins to mammalian cells that will also offer protection against β-lactamase-induced hydrolysis, nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) have been employed to study the inclusion complexes formed in aqueous solution between designed CD derivatives and two aminopenicillins, ampicillin and amoxicillin, and two antistaphylococcal penicillins, methicillin and oxacillin. Anionic and cationic thioether-substituted-β- and -γCD derivatives were thus synthesized and compared with the neutral, parent CDs for complexation with the penicillins. The synthesized derivatives were shown to present ∼20% elongated cavity space in solution. Moreover, the cationic ones are >98% protonated at physiological pH. The most efficient host was the positively charged octakis[6-(2-aminoethylthio)-6-deoxy]-γ-CD (γCys) that formed the strongest complex with oxacillin (Kb ∼1700M-1) in an enthalpically and entropically favorable process (ΔHb=-10.5kJ/mol,TΔSb=8.0kJ/mol). In vitro biological tests demonstrated that γCys reduces 2.3-fold the rate of hydrolysis of oxacillin in the presence of oxa-1 β-lactamase while displaying cell crossing capability and efficient internalization into macrophages as well as a sufficiently safe cytotoxicity profile. Overall, γCys could be considered as a promising vehicle for protection and delivery of oxacillin.

By searching the Integrall integron and GenBank databases, a novel open reading frame (ORF) of 51 nucleotides (nts) (ORF-17) overlapping the previously described ORF-11 was identified within the attI1 site in virtually all class 1 integrons. Using a set of isogenic plasmid constructs carrying a single gene cassette ( bla GES-1 ) and possessing a canonical translation initiation region, we found that ORF-17 contributes to GES-1 expression.

OBJECTIVES 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. METHODS 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. RESULTS 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 qnrS1. 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 blaCMY-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. CONCLUSIONS The long-term maintenance of FQ- and ESC-resistant clones harbouring topoisomerase mutations and a blaCMY-2-IncI1/ST65 plasmid in canine commensal flora after prolonged antimicrobial use may contribute to the dissemination of multidrug resistance.

Carbapenemase-producing Klebsiella pneumoniae strains (CP-Kps) are currently among the most important nosocomial pathogens. An observational study was conducted during 2009 to 2010 in two hospitals located in a high-prevalence area (Athens, Greece). The aims were (i) to evaluate the clinical outcome of patients with CP-Kp bloodstream infections (BSIs), (ii) to identify predictors of mortality, and (iii) to evaluate the various antibiotic schemes employed. A total of 205 patients with CP-Kp BSIs were identified: 163 (79.5%) were infected with KPC or KPC and VIM, and 42 were infected with VIM producers. For definitive treatment, 103 patients received combination therapy (two or more active drugs), 72 received monotherapy (one active drug), and 12 received therapy with no active drug. The remaining 18 patients died within 48 h after the onset of bacteremia. The all-cause 28-day mortality was 40%. A significantly higher mortality rate was observed in patients treated with monotherapy than in those treated with combination therapy (44.4% versus 27.2%; P = 0.018). The lowest mortality rate (19.3%) was observed in patients treated with carbapenem-containing combinations. In the Cox proportion hazards model, ultimately fatal disease (hazards ratio [HR], 3.25; 95% confidence interval [CI], 1.51 to 7.03; P = 0.003), the presence of rapidly fatal underlying diseases (HR, 4.20; 95% CI, 2.19 to 8.08; P < 0.001), and septic shock (HR, 2.15; 95% CI, 1.16 to 3.96; P = 0.015) were independent predictors of death. Combination therapy was strongly associated with survival (HR of death for monotherapy versus combination, 2.08; 95% CI, 1.23 to 3.51; P = 0.006), mostly due to the effectiveness of the carbapenem-containing regimens.

The CMY-type enzymes are the most frequent acquired class C β-lactamases ([1][1]). Spread of the respective genes is mediated mostly by IncI and IncA/C plasmids ([2][2][–][3][4][4]). bla CMY-carrying ColE1 replicons have also been reported ([5][5]). Here, we describe a CMY-4-encoding IncQ plasmid

The objective of the study is to report a multidrug-resistant outbreak of Providencia stuartii that occurred in inpatients in the Athens area in 2012 resulting from a very successful transmissible A/C multidrug-resistant plasmid. Thirteen multidrug-resistant P. stuartii clinical isolates from 5 hospitals were studied. Molecular typing was performed by pulsed-field gel electrophoresis. Antibiotic resistance genes and their genetic surround were detected by PCR and sequencing. Plasmid analysis included conjugation experiments using liquid cultures, sizing by S1 digestion, and incompatibility replicon typing by PCR. Isolates were grouped into 2 distinct clonal types A and B, exhibiting similarity less than 70%. Isolates of type A were recovered from patients hospitalized in 4 different hospitals with no obvious epidemiological linkage, while isolates of type B were recovered from patients treated in a single hospital. Both clonal types harbored a conjugative plasmid of 130 bp and IncA/C replicon type carrying 5 β-lactamase genes bla(SHV-5), bla(VEB-1), bla(VIM-1), bla(OXA-10), and bla(TEM-1) and aminoglycosides resistant determinants. All β-lactamase genes were included in stable structures as IS26, IS1999, and In-e541. The current plasmid seemed to have many common determinants with previously reported plasmids derived from P. stuartii and Proteus mirabilis clinical isolates and exhibited the ability to circulate in nosocomial bacterial populations.

Klebsiella pneumoniae strains co-producing klebsiella pneumoniae carbapenemase (KPC) and verona integron-encoded metallo-beta-lactamase (VIM) are frequently isolated in Greece and have also occurred in other European countries. Conventional combined disc tests exhibit low sensitivity against these emerging pathogens. We have evaluated modifications of the KPC/Metallo-β-Lactamase Confirmation kit (ROSCO) exhibiting high diagnostic value against KPC, VIM and KPC + VIM producers. The key changes were the inclusion of additional combined tablets containing meropenem plus two inhibitors (dipicolinic acid (1000 μg per tablet) for metallo-β-lactamases and a boronic acid derivative for KPCs) and the replacement of aminophenylboronic acid by phenylboronic acid (400 μg per tablet).

Porins are represented in large amounts in outer membrane and form non-specific diffusion channels, allowing small, polar molecules (

Plasmid-acquired carbapenemases in Enterobacteriaceae, which were first discovered in Europe in the 1990s, are now increasingly being identified at an alarming rate. Although their hydrolysis spectrum may vary, they hydrolyse most β-lactams, including carbapenems. They are mostly of the KPC, VIM, NDM and OXA-48 types. Their prevalence in Europe as reported in 2011 varies significantly from high (Greece and Italy) to low (Nordic countries). The types of carbapenemase vary among countries, partially depending on the cultural/population exchange relationship between the European countries and the possible reservoirs of each carbapenemase. Carbapenemase producers are mainly identified among Klebsiella pneumoniae and Escherichia coli, and still mostly in hospital settings and rarely in the community. Although important nosocomial outbreaks with carbapenemase-producing Enterobacteriaceae have been extensively reported, many new cases are still related to importation from a foreign country. Rapid identification of colonized or infected patients and screening of carriers is possible, and will probably be effective for prevention of a scenario of endemicity, as now reported for extended-spectrum β-lactamase (mainly CTX-M) producers in all European countries.

CMY-30, a naturally occurring class C β-lactamase differing from the Citrobacter freundii-derived CMY-2 by a Val211Gly substitution in the Ω-loop, exhibits increased hydrolytic efficiency against ceftazidime and cefotaxime. Kinetic constants of CMY-2 and CMY-30 against the latter substrates suggested that the improved efficiency of the Gly211 variant was due to an increase in kcat. The structural basis of the increased turn-over rates of oxyimino-cephalosporins caused by Val211Gly was studied using 5 ns molecular dynamics simulations of CMY-2 and CMY-30 in their free forms and in covalent complexes with ceftazidime (acyl-enzyme) as well as a boronic acid analogue of ceftazidime (deacylation transition state). Analysis of thermal factors indicated that Val211Gly increased the flexibility of the Ω-loop/H7-helix and the Q120-loop formed by amino acids 112–125, and also altered the vibrations of the H10-helix/R2-loop. Structural elements containing the catalytic residues remained relatively rigid except Tyr150 in acyl-enzyme species. Regions exhibiting altered flexibility due to the substitution appear to move in a concerted manner in both enzymes. This movement was more intense in CMY-30 and also at directions different to those observed for CMY-2. Additionally, it appeared that the Val211Gly increased the available space for the accommodation of the R1 side chain of ceftazidime. These findings are likely associated with the significantly increased vibrations of the bound compounds observed in CMY-30 complexes. Therefore, the extended spectrum properties of CMY-30 seem to arise through a complex process implicating changes in protein movement and in the mode of substrate accommodation. Proteins 2011;. © 2011 Wiley-Liss, Inc.

We isolated a clinical Escherichia coli strain with an antimicrobial resistance phenotype characteristic for the expression of an AmpC beta-lactamase. Molecular methods revealed a novel, plasmid-localized variant of CMY-2 with a substitution of valine 231 for serine (V231S), which was designated CMY-42. Like the CMY-2-like AmpC beta-lactamase CMY-30, carrying the substitution V231G, CMY-42 displayed increased activity toward expanded spectrum cephalosporins. This finding supports the hypothesis that a bulky side chain at position 231 (Ambler's position 211) may pose a steric clash with certain cephalosporins hindering the access of the AmpC beta-lactamase; however, additional phenomena may account for the observed hydrolytic properties.

In GES-type β-lactamases, positions 104 and 170 are occupied by Glu or Lys and by Gly, Asn, or Ser, respectively. Previous studies have indicated an important role of these amino acids in the interaction with β-lactams, although their precise role, especially that of residue 104, remains uncertain. In this study, we constructed GES-1 (Glu104, Gly170), GES-2 (Glu104, Asn170), GES-5 (Glu104, Ser170), GES-6 (Lys104, Ser170), GES-7 (Lys104, Gly170), and GES-13 (Lys104, Asn170) by site-specific mutagenesis and compared their hydrolytic properties. Isogenic comparisons of β-lactam resistance levels conferred by these GES variants were also performed. Data indicated the following patterns: (i) Lys104-containing enzymes exhibited enhanced hydrolysis of oxyimino-cephalosporins and reduced efficiency against imipenem in relation to enzymes possessing Glu104, (ii) Asn170-containing enzymes showed reduced hydrolysis rates of penicillins and older cephalosporins, (iii) Ser170 enabled GES to hydrolyze cefoxitin efficiently, and (iv) Asn170 and Ser170 increased the carbapenemase character of GES enzymes but reduced their activity against ceftazidime. Molecular dynamic simulations of GES apoenzyme models, as well as construction of GES structures complexed with cefoxitin and an achiral ceftazidime-like boronic acid, provided insights into the catalytic behavior of the studied mutants. There were indications that an increased stability of the hydrogen bonding network of Glu166-Lys73-Ser70 and an altered positioning of Trp105 correlated with the substrate spectra, especially with acylation of GES by imipenem. Furthermore, likely effects of Ser170 on GES interactions with cefoxitin and of Lys104 on interactions with oxyimino-cephalosporins were revealed. Overall, the data unveiled the importance of residues 104 and 170 in the function of GES enzymes.

Two CMY-2 derivatives, CMY-31 (Gln 215 →Arg) from Salmonella enterica serotype Newport and CMY-36 (Ala 77 →Cys and Gln 193 →Glu) from Klebsiella pneumoniae , were characterized. Both cephalosporinases functionally resembled CMY-2. bla CMY alleles occurred as parts of a putative transposon comprising IS Ecp1B and a Citrobacter freundii -derived sequence carried by ColE1-like plasmids similar to CMY-5-encoding pTKH11 from Klebsiella oxytoca .

OBJECTIVES Global dissemination of KPC-type carbapenemases is mainly associated with the spread of high-risk clones of Klebsiella pneumoniae and of KPC-encoding plasmids. In this study, we explored the population structure of KPC-encoding plasmids from the recent epidemics of KPC-producing K. pneumoniae (KPC-Kp) in Greece and Italy, the two major European endemic settings. METHODS Thirty-four non-replicate clinical strains of KPC-Kp representative of the early phases (2008-11) of the Greek (n = 22) and Italian (n = 12) epidemics were studied. Isolates were typed by MLST, and blaKPC-carrying plasmids were characterized by S1 profiling, PCR-based replicon typing and RFLP. Transfer experiments by conjugation or transformation were carried out with Escherichia coli recipients. Eleven plasmids, representative of all different restriction profiles, were completely sequenced. RESULTS The representative Greek strains belonged to 14 sequence types (STs), with a predominance of ST258. The representative Italian strains belonged to three STs, with a predominance of clonal complex 258 (ST258, ST512). The 34 strains carried plasmids of variable size (78-166 kb), either with blaKPC-2 or blaKPC-3 gene embedded in a Tn4401a transposon. Plasmids from Greek strains were mostly of a single RFLP type (A) and resembled the archetypal pKpQIL KPC-encoding plasmid, while plasmids from Italian strains belonged to a more heterogeneous population, showing five RFLP profiles (A, C-F). Types A and C resembled pKpQIL or deletion derivatives thereof, while types D-F included plasmids with hybrid structures between pKpQIL, pKPN3 and pKPN101-IT. CONCLUSIONS pKpQIL-like plasmids played a major role in the dissemination of blaKPC in Greece and Italy, but evolved with different dynamics in these endemic settings.

Proteus mirabilis NO-051/03, representative of a multidrug-resistant clone expressing the CMY-16 AmpC-type β-lactamase and circulating in Europe since 2003, was sequenced by a MiSeq platform using a paired-end approach. The genome was assembled in 100 scaffolds with a total length of 4,197,318 bp. Analysis of the draft genome sequence revealed the presence of several acquired resistance determinants to β-lactams, aminoglycosides, phenicols, tetracyclines, trimethoprim, and sulfonamides, of one plasmid replicon, and of a type I-E clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) adaptive immune system.

VIM-producing Klebsiella pneumoniae isolates are usually associated with high MICs to carbapenems. Preclinical studies investigating the pharmacokinetic-pharmacodynamic (PK-PD) characteristics of carbapenems against these isolates are lacking. The in vitro antibacterial activity of meropenem against one WT and three VIM-producing K. pneumoniae clinical isolates (median MICs 0.031, 8, 16 and 128 mg l(-1)) was studied in a dialysis-diffusion PK-PD model and verified in a thigh infection neutropenic animal model by testing selected strains and exposures. The in vitro PK-PD target associated with bactericidal activity was estimated and the target attainment for different dosing regimens was calculated with Monte Carlo analysis. The in vitro model was correlated with the in vivo data, with log(10)CFU/ml reduction of 2 for the WT (MIC 0.031 mg l(-1)) isolates, with %fT >MIC 25 and 100 %, respectively. The in vitro bactericidal activity for all isolates was associated with 40 % fT>MIC and attained in >90 % of cases with the standard 1 g q8 0.5 h infusion dosing regimen only for isolates with MICs up to 1 mg l(-1). For isolates with MICs of 2-8 mg l(-1), prolonged infusion regimens (4 h infusion q8 or 2 h infusion q4) of standard (1 g) and higher (2 g) doses or continuous infusion regimens (3-6 g) are required. For isolates with a MIC of 16 mg l(-1) the unconventional dosing regimen of 2 g as 2 h infusion q4 or 12 g continuous infusion will be required. Prolonged and continuous infusion regimens of meropenem may increase efficacy against VIM-producing K. pneumoniae isolates.

ACC-4, an omega loop mutant (Val 211 →Gly) of the Hafnia alvei -derived cephalosporinase ACC-1, was encoded by an Escherichia coli plasmid. The genetic environment of bla ACC-4 shared similarities with plasmidic regions carrying bla ACC-1 . Kinetics of β-lactam hydrolysis and levels of resistance to β-lactams showed that ACC-4 was more effective than ACC-1 against expanded-spectrum cephalosporins.

A novel class A β-lactamase (SCO-1) encoded by an 80-kb self-transferable plasmid from Escherichia coli is described. The interaction of SCO-1 with β-lactams was similar to that of the CARB-type enzymes. Also, SCO-1 exhibited a 51% amino acid sequence identity with the RTG subgroup of chromosomal carbenicillinases (RTG-1, CARB-5, and CARB-8).

The in-vivo activity of imipenem against VIM-1-producing Klebsiella pneumoniae (VPKP) was assessed in a thigh infection model in neutropenic mice. Animals were infected with three VPKP isolates (imipenem MICs 2, 4 and 32 mg/L, respectively) and a susceptible clinical isolate (MIC 0.125 mg/L) that did not produce any β-lactamase with broad-spectrum activity. Bacterial density at the site of infection was determined after imipenem treatment (30 and 60 mg/kg every 2 h for 24 h). The log10 reduction in CFU/thigh was greatest for the wild-type isolate, intermediate for the two imipenem-susceptible VPKP isolates, and lowest for the imipenem-resistant VPKP isolate. Whilst in-vivo imipenem activity appeared reduced against in-vitro susceptible VIM-1 producers compared with a VIM-1-negative control, an increased drug dosage could moderate this reduction.

Genes conferring simultaneous resistance to different classes of antimicrobials, confer a selective advantage to the host, particularly when those corresponding antibiotics are administered. Multiple resistance genes clustered within the same genetic locus (resistance island) can be transferred en bloc to other organisms. In this chapter we review novel multidrug resistance islands recently described in Salmonella.

Objectives: Surveillance data concerning antimicrobial susceptibilities of Neisseria gonorrhoeae isolated in Greece during the 11 year period 1994–2004 are presented. Methods: Antimicrobial susceptibilities of all gonococcal isolates received by the Greek National Reference Center for N. gonorrhoeae during the study period were determined in terms of MICs using Etest. Trends in yearly isolation frequencies by susceptibility category were estimated for defining significant changes in overall susceptibility figures. Results: Cefotaxime and spectinomycin retained undiminished activity against all isolates throughout the study period. High rates of resistance and intermediate susceptibilities were noticed for penicillin, tetracycline and erythromycin, and even for norfloxacin and ciprofloxacin. A substantial portion (16.5%) of the gonococcal samples consisted of multiresistant strains exhibiting resistance to two or more agents of different antibiotic classes. Although annual rates of low-level chromosomal resistance decreased, high-level resistance owing to the presence of penicillin- and tetracycline-resistance plasmids increased. Fluoroquinolone resistance also showed a significant increasing trend after 1996, reaching a peak rate of 11.3% in 2004. Conclusion: Third-generation cephalosporins and spectinomycin should be considered as first-choice drugs for the empirical treatment of gonorrhoea in Greece.

The nucleotide sequence of pKP1433 (55,417 bp), a bla KPC-2 -carrying plasmid from Klebsiella pneumoniae sequence type 340, was determined. pKP1433 displayed extensive sequence and structural similarities with the IncN plasmids possessing the KPC-2-encoding Tn 4401b isoform. However, the replication, partitioning, and stability of pKP1433 were determined by sequences related to diverse non-IncN plasmids.

Two clinical isolates, an Escherichia coli and a Klebsiella pneumoniae, with decreased susceptibility to carbapenems were studied. This phenotype was associated with production of novel GES/IBC variant β-lactamases, designated GES-3 (from E. coli) and GES-4 (from K. pneumoniae), exhibiting carbapenemase activity. Both enzymes possessed Ser at Ambler's position 170 instead of Gly found in the β-lactamases GES-1 and IBC-1 that lack carbapenemase activity. Additionally, position 104 in GES-4 was occupied by a Lys as in IBC-1. blaGES-3 and blaGES-4 occurred as gene cassettes in the variable regions of class 1 integrons carried by plasmids. The structure of the GES-4-encoding integron was similar to that of previously described IBC-1 integrons. The GES-3-encoding integron was, most likely, truncated at the 3′ conserved segment.

A class 1 integron, In111, carried by a self-transferable plasmid from an Escherichia coli clinical strain was characterized. The variable region of In111 constituted an array of gene cassettes encoding the extended-spectrum beta-lactamase IBC-1, the aminoglycoside-modifying enzymes AAC(6')-Ib and ANT(3")-Ia, dihydrofolate reductase I and a putative polypeptide (SMR-2) sharing similarity with the Qac transporters. Transcription of the gene cassettes was driven by a hybrid-type P1 promoter located in a typical 5' conserved segment (CS). The 3'CS included sulI, qacEDelta1, orf5 and orf6. In111 was bounded on the right by an inversely oriented IRt. The 5'CS was preceded by an intact IS26 element followed by an aphA1 gene.

Seventeen Klebsiella pneumoniae clinical isolates carrying the bla VIM-1 metallo-β-lactamase gene were collected in the intensive care units of three hospitals in Athens, Greece, in 2002. They exhibited various carbapenem resistance levels (Etest MICs of imipenem ranged from 4 to 32 μg/ml). All isolates gave positive results by the imipenem-EDTA synergy Etest. The isolates were classified into four main types by pulsed-field gel electrophoresis; the majority of the isolates (5 and 10 isolates) belonged to two types. The bla VIM-1 gene cassette was part of the variable region of a class 1 integron that also included aac6 , dhfrI , and aadA . This structure was carried by transferable plasmids.

A Salmonella enterica serotype Cubana isolate exhibiting resistance to most β-lactam antibiotics, including oxyimino-cephalosporins and imipenem, was isolated from a 4-year-old boy with gastroenteritis in Maryland. β-Lactam resistance was mediated by a conjugative plasmid that encoded KPC-2, a class A carbapenemase previously found in a Klebsiella pneumoniae isolate from the Maryland area as well. Sequence analysis of the flanking regions indicated a potential association of bla KPC-2 with mobile structures.

Oxacillin resistance was examined in 258 coagulase-negative staphylococci from Greek hospitals. mecA DNA was detected in 168 isolates, which were also resistant to oxacillin by agar dilution and disk diffusion, according to the current NCCLS breakpoints. Both methods exhibited a relatively low specificity misclassifying 21 and 19 of the 90 mecA-negative isolates respectively as oxacillin resistant. In contrast, an anti-PBP 2a latex agglutination test, applied after induction by oxacillin, correctly classified 163 mecA-positive (sensitivity 97%) and 88 mecA-negative isolates (specificity 97.7%).