Ointments containing Ceratothoa oestroides extract: Evaluation of their healing potential in the treatment of diabetic foot ulcers.

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.

attI1-Located Small Open Reading Frames ORF-17 and ORF-11 in a Class 1 Integron Affect Expression of a Gene Cassette Possessing a Canonical Shine-Dalgarno Sequence.

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 (blaGES-1) and possessing a canonical translation initiation region, we found that ORF-17 contributes to GES-1 expression.

Characterization of KPC-encoding plasmids from two endemic settings, Greece and Italy.

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.

Carbapenemase-producing Klebsiella pneumoniae bloodstream infections: lowering mortality by antibiotic combination schemes and the role of carbapenems.

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.

Successful control of a neonatal outbreak caused mainly by ST20 multidrug-resistant SHV-5-producing Klebsiella pneumoniae, Greece.

BACKGROUND: Extended spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-Kp) infection can cause significant morbidity and mortality in neonates. We investigated a nosocomial ESBL-Kp outbreak in a neonatal intensive care unit (NICU) of the University Hospital of Larissa (UHL), Central Greece. METHODS: A total of sixty-four ESBL-Kp were studied; twenty six isolates were recovered from the NICU and were compared with thirty-eight randomly selected isolates from different wards of the hospital during the period March- December 2012. All isolates were characterized by antimicrobial susceptibility testing, ESBL-production by double-disk synergy test, molecular typing using BOX-PCR, whereas selected isolates were further characterized by beta lactamase and virulence gene content, multilocus sequence typing and phylogenetic analysis. All neonates affected by ESBL-Kp were put under strict contact isolation, along with appropriate infection control measures. RESULTS: The outbreak strain of ST20 multidrug-resistant SHV-5-producing K. pneumoniae was identified in all infected (n = 13) and three colonized neonates. A novel ST (ST1114) was also identified among SHV-5 producers (n = 10) recovered from nine colonized infants, but it was not related with ST20. Both STs were identified only in the NICU and not in other wards of the hospital. No ESBL-Kp were isolated from the hands of the nursing staff and the environment. Although we were not able to identify the source of the outbreak, no ESBL-Kp were isolated in the NICU after this period and we assumed that the outbreak was successfully controlled. All neonates received parenteral nutrition and most of them were delivered by caesarean section and showed low gestational age (<32 weeks) and low birth weights (<1500 g). CONCLUSION: According to our knowledge, this is the first description of an outbreak of multidrug-resistant SHV-5 producing K. pneumoniae assigned to ST20.

Carbapenem-resistant Klebsiella pneumoniae: the role of plasmids in emergence, dissemination, and evolution of a major clinical challenge.

INTRODUCTION: Klebsiella pneumoniae is a major agent of healthcare-associated infections and a cause of some community-acquired infections, including severe bacteremic infections associated with metastatic abscesses in liver and other organs. Clinical relevance is compounded by its outstanding propensity to evolve antibiotic resistance. In particular, the emergence and dissemination of carbapenem resistance in K. pneumoniae has posed a major challenge due to the few residual treatment options, which have only recently been expanded by some new agents. The epidemiological success of carbapenem-resistant K. pneumoniae (CR-Kp) is mainly linked with clonal lineages that produce carbapenem-hydrolyzing enzymes (carbapenemases) encoded by plasmids. AREAS COVERED: Here, we provide an updated overview on the mechanisms underlying the emergence and dissemination of CR-Kp, focusing on the role that plasmids have played in this phenomenon and in the co-evolution of resistance and virulence in K. pneumoniae. EXPERT OPINION: CR-Kp have disseminated on a global scale, representing one of the most important contemporary public health issues. These strains are almost invariably associated with complex multi-drug resistance (MDR) phenotypes, which can also include recently approved antibiotics. The heterogeneity of the molecular bases responsible for these phenotypes poses significant hurdles for therapeutic and diagnostic purposes.

Interactions of oximino-substituted boronic acids and ß-lactams with the CMY-2-derived extended-spectrum cephalosporinases CMY-30 and CMY-42.

CMY-30 and CMY-42 are extended-spectrum (ES) derivatives of CMY-2. ES characteristics are due to substitutions of Gly (CMY-30) and Ser (CMY-42) for Val211 in the Ω-loop. To characterize the effects of 211 substitutions, we studied the interactions of CMY-2, -30, and -42 with boronic acid transition state inhibitors (BATSIs) resembling ceftazidime and cefotaxime, assessed thermal stability of the enzymes in their free forms and in complexes with BATSIs and oximino-ß-lactams, and simulated, using molecular dynamics (MD), the CMY-42 apoenzyme and the CMY-42 complexes with ceftazidime and the ceftazidime-like BATSI. Inhibition constants showed that affinities between CMY-30 and CMY-42 and the R1 groups of BATSIs were lower than those of CMY-2. ES variants also exhibited decreased thermal stability either as apoenzymes or in covalent complexes with oximino compounds. MD simulations further supported destabilization of the ES variants. Val211Ser increased thermal factors of the Ω-loop backbone atoms, as previously observed for CMY-30. The similar effects of the two substitutions seemed to be due to a less-constrained Tyr221 likely inducing concerted movement of elements at the edges of the active site (Ω-loop-Q120 loop-R2 loop/H10 helix). This inner-protein movement, along with the wider R1 binding cleft, enabled intense vibrations of the covalently bound ceftazidime and ceftazidime-like BATSIs. Increased flexibility of the ES enzymes may assist the productive adaptation of the active site to the various geometries of the oximino substrates during the reaction (higher frequency of near-attack conformations).

Carbapenemase-producing Pseudomonas aeruginosa from central Greece: molecular epidemiology and genetic analysis of class I integrons.

BACKGROUND: Multidrug-resistant Pseudomonas aeruginosa is a serious challenge for antimicrobial therapy of nosocomial infections, as it possesses several mechanisms of antimicrobial resistance. In Central Greece, a sudden increase of infections caused by carbapenem-resistant P. aeruginosa was observed during 2011, indicating the need for further analysis. METHODS: Five-hundred and sixty-eight P. aeruginosa isolates were collected consecutively during an 8-month period in 2011 from inpatients treated in three hospitals in the Thessaly region (1,000,000 habitants) of Greece. Carbapenem-resistant P. aeruginosa (n = 284) were characterized by antimicrobial susceptibility testing and ß-lactamase content, and the genetic relatedness of carbapenemase-producing isolates was assessed by BOX-PCR, multilocus sequence typing, and eBURST analysis. Mapping of the class I integrons of Verona integron-encoded metallo-ß-lactamase (VIM)-carrying isolates was also performed, and clinical data of the VIM producers were reviewed. RESULTS: Eighty (14.1%) out of the 568 P. aeruginosa isolates recovered from clinical specimens were VIM producers. Multilocus sequence typing revealed high prevalence of the international clones ST111 and ST235 among blaVIM-2- and blaVIM-4-positive isolates, respectively. blaVIM-17 was identified in an isolate of a novel sequence type (ST1457). blaVIM gene cassettes were carried by five distinct class I integrons, including two novel ones. CONCLUSIONS: Since the first report of VIM-producing P. aeruginosa in 2000, this microorganism still remains among the most prevalent multidrug resistant pathogens in Greece. The spread of VIM-producers belonging to the most common international clones (ST111 and ST235), the spread of integrons of divergent structures, and the emergence of novel integrons underscore their ongoing evolution.

Diversity of Bacterial Clones and Plasmids of NDM-1 Producing Escherichia coli Clinical Isolates in Central Greece.

The objective of the present study was to genetically characterize ten NDM-1 producing Escherichia coli isolates, recovered from patients in a hospital in Central Greece during the period 2017 to 2021.The isolates were studied by whole genome sequencing to obtain multi-locus sequencing typing (MLST), identification of blaNDM1-environment, resistome and plasmid content. MLST analysis showed the presence of eight sequence types: ST46* (two isolates), ST46, ST744, ST998, ST410, ST224, ST4380, ST683 and ST12 (one isolate each). Apart of the presence of blaNDM-1, the isolates carried a combination of various to ß-lactams encoding resistance genes: blaTEM-1B, blaCTX-15, blaOXA-1, blaVIM-1, blaSHV-5, blaOXA-16, blaOXA-10 and blaVEB-1. Additionally, plurality of resistance genes to aminoglycosides, macrolides, rifamycin, phenicols, sulfonamides and tetracycline was detected. The presence of multiple replicons was observed, with predominance of IncFII and IncFIB. Analysis of blaNDM-1 genetic environment of the isolates showed that seven had 100% identity with the pS-3002cz plasmid (Accession Number KJ 958927), two with the pB-3002cz plasmid (Accession Number KJ958926) and one with the pEc19397-131 plasmid (Accession Number MG878866). Τhis latter plasmid was derived by the fusion of two, previously identified, plasmids, pAMPD2 and pLK75 (Accession Numbers CP078058 and KJ440076, respectively). The diversity of clones and plasmids of NDM-1 producing E. coli isolated from patients in Greece indicates a continuous horizontal gene transfer.

Molecular Characterization and Prevalence of Antimicrobial-Resistant Escherichia coli Isolates Derived from Clinical Specimens and Environmental Habitats.

Antibiotic-resistant bacteria (ARB) are present in wastewaters as their elimination during treatment in wastewater treatment plants (WWTPs) is often impossible. Water plays an important role in the spread of these microorganisms among humans, animals and the environment. This study aimed to assess the antimicrobial resistance patterns, resistance genes and molecular genotypes by means of phylogenetic groups of E. coli isolates in aquatic habitats, including sewage and receiving water bodies, as well as clinical settings in the Boeotia regional district of Greece. The highest resistance rates among both environmental and clinical isolates were observed to be for penicillins, ampicillin and piperacillin. Resistance patterns related to extended spectrum ß-lactamases (ESBL) production and ESBL genes were also detected in both environmental and clinical isolates. Phylogenetic group B2 was predominant in clinical settings and the second most frequent among wastewaters, whereas group A was dominant in all environmental isolates. In conclusion, the studied river water and wastewaters may serve as reservoirs of resistant E. coli isolates that pose potential threats to both human and animal health.

First Detection and Molecular Characterization of Pseudomonas aeruginosa blaNDM-1 ST308 in Greece.

The objective of the present study is to report the detection and the molecular characterization of nine blaNDM-1-positive Pseudomonas aeruginosa isolates, all of which belonged to the epidemic high-risk international clone ST308, and all were isolated from patients in a tertiary care hospital in Central Greece from May to July 2023.The isolates were characterized by whole genome sequencing to obtain multi-locus sequencing typing (MLST) and identify the blaNDM1-environment and resistome and virulence genes content. In silico MLST analysis showed that all isolates belonged to the high-risk ST308 international clone. All strains possessed 22 different genes, encoding resistance to various antimicrobial agents. Whole genome sequencing revealed that the blaNDM-1 was chromosomally located within the integrative and conjugative element ICETn43716385 and that it was part of one cassette along with two other resistance genes, floR and msrE. Two additional resistance cassettes were also found in the genome, which included the arrays of aph(6)-Id, aph(3″)-Ib, floR, sul2 and aadA10, qnrVC1, aac(3)-Id, dfrB5, aac(6')-II. Additionally, the strains possessed various virulence genes, e.g., aprA, exoU, lasA, lasB, toxA, and estA. All of the isolates shared identical genomes, which showed 98% similarity with the P. aeruginosa ST308 genome (acc. no CP020703), previously reported from Singapore. To our knowledge, this is the first report of ST308 blaNDM-1-positive P. aeruginosa isolation in Europe, which indicates the transmission dynamics of this high-risk clone.

The Molecular Characterization of blaNDM-1-Positive Acinetobacter baumannii Isolated in Central Greece.

The objective of the present study is to report the detection and the molecular characterization of nine blaNDM-1-positive Acinetobacter baumannii isolates, which were isolated from patients in a tertiary care hospital in Central Greece from December 2022 to August 2023. The isolates were characterized by whole genome sequencing to obtain Pasteur multilocus sequencing typing (MLST) and to identify the blaNDM-1-environment, resistome, and virulence genes content. In silico MLST analysis showed that the isolates belonged to four different clones (STs 160, 2, 85, and 2493). All strains, apart from the blaNDM-1-gene, possessed at least eight different genes, encoding resistance to various antimicrobial agents. Whole genome sequencing revealed two different structures of the blaNDM-1 environment. The first, detected in ST160 strain, was identical with the Tn125, whereas the second, found in STs 2, 85, and 2493 was associated with Tn7382. To our knowledge, after a sole strain reported in 2016 and imported by a patient hospitalized in a Libyan hospital, this is the first report of the emergence of polyclonal blaNDM-1-positive Acinetobacter baumannii in Greece. Our findings re-emphasize the need to apply diligent surveillance protocols in order to limit the horizontal transfer of the blaNDM-1 gene to other A. baumannii clones or to other recipient strains.

Metal-binding cyclodextrins: Synthesis and complexation with Zn2+ and Ga3+ cations towards antimicrobial applications.

Highly resistant bacteria producing metallo-ß-lactamases (MBLs) to evade ß-lactam antibiotics, constitute a major cause of life-threatening infections world-wide. MBLs exert their hydrolytic action via Zn2+ cations in their active center. Presently, there are no approved drugs to target MBLs and combat the associated antimicrobial resistance (AMR). Towards this issue, we have prepared a family of cyclodextrins substituted with iminodiacetic acid (IDA) on their narrow side, while the wider side is either unmodified or per-2,3-O-methylated. The molecules form strong coordination complexes with Zn2+ or Ga3+ cations in aqueous solution. Free and metal-complexed compounds have been thoroughly characterized regarding structures, pH-dependent ionization states, distribution of species in solution, pKa values and metal-binding constants. At neutral pH the multi-anionic hosts bind up to four Zn2+ or Ga3+ cations. In vitro, 50 µΜ of the compounds achieve complete re-sensitization of MBL-producing Gram-negative clinical bacterial strains resistant to the carbapenems imipenem and meropenem. Moreover, the radioactive complex [67Ga]Ga-ß-IDACYD prepared, displays high radiochemical purity, sufficient stability both overtime and in the presence of human plasma apo-transferrin, thus providing an invaluable tool for future biodistribution and pharmacokinetic studies of ß-IDACYDin vivo, prerequisites for the development of therapeutic protocols.

Ciprofloxacin-resistant Escherichia coli in Central Greece: mechanisms of resistance and molecular identification.

BACKGROUND: Fluoroquinolone resistant E. coli isolates, that are also resistant to other classes of antibiotics, is a significant challenge to antibiotic treatment and infection control policies. In Central Greece a significant increase of ciprofloxacin-resistant Escherichia coli has occurred during 2011, indicating the need for further analysis. METHODS: A total of 106 ciprofloxacin-resistant out of 505 E. coli isolates consecutively collected during an eight months period in a tertiary Greek hospital of Central Greece were studied. Antimicrobial susceptibility patterns and mechanisms of resistance to quinolones were assessed, whereas selected isolates were further characterized by multilocus sequence typing and ß-lactamase content. RESULTS: Sequence analysis of the quinolone-resistance determining region of the gyrA and parC genes has revealed that 63% of the ciprofloxacin-resistant E. coli harbored a distinct amino acid substitution pattern (GyrA:S83L + D87N; ParC:S80I + E84V), while 34% and 3% carried the patterns GyrA:S83L + D87N; ParC:S80I and GyrA:S83L + D87N; ParC:S80I + E84G respectively. The aac (6')-1b-cr plasmid-mediated quinolone resistance determinant was also detected; none of the isolates was found to carry the qnrA, qnrB and qnrS.Genotyping of a subset of 35 selected ciprofloxacin-resistant E. coli by multilocus sequence typing has revealed the presence of nine sequence types; ST131 and ST410 were the most prevalent and were exclusively correlated with hospital and health care associated infections, while strains belonging to STs 393, 361 and 162 were associated with community acquired infections. The GyrA:S83L + D87N; ParC:S80I + E84V substitution pattern was found exclusively among ST131 ciprofloxacin-resistant E. coli. Extended-spectrum ß-lactamase-positive ST131 ciprofloxacin-resistant isolates produced CTX-M-type enzymes; eight the CTX-M-15 and one the CTX-M-3 variant. CTX-M-1 like and KPC-2 enzymes were detected in five and four ST410 ciprofloxacin-resistant E. coli isolates, respectively. CONCLUSIONS: Our findings suggest that, ST131 and ST410 predominate in the ciprofloxacin resistant E. coli population.

Direct Colorimetry of Imipenem Decomposition as a Novel Cost-Effective Method for Detecting Carbapenemase-Producing Enterobacteria.

In the absence of a molecule that would collectively inhibit both metallo-ß-lactamases and serine-reactive carbapenemases, containment of their genes is the main weapon currently available for confronting carbapenem 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 ß-lactamases from the four molecular classes. The effects of various buffers on reaction mixtures 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 66 selected enterobacteria, including 50 CPE as well as 16 carbapenemase-negative strains overexpressing other ß-lactamases. The development of the yellow color was specific for carbapenemase-containing enzyme preparations, and the maximum intensity was achieved in acidic or unbuffered conditions in the presence of zinc. When applied on bacterial cell suspensions, the assay could detect CPE with 98% 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 exhibiting high accuracy in detecting CPE. Therefore, it should be considered for screening purposes after further clinical evaluation. IMPORTANCE Currently, the spread of multidrug-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 ß-lactamases is of vital importance. Herein, we developed a novel method, based on a previously undescribed phenomenon, that can detect CPE with few reagents by direct colorimetry of bacterial suspensions and imipenem/cilastatin mixtures.

Effects of the Val211Gly substitution on molecular dynamics of the CMY-2 cephalosporinase: implications on hydrolysis of expanded-spectrum cephalosporins.

CMY-30, a naturally occurring class C ß-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 k(cat). 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.

Analysis of emergence of quinolone-resistant gonococci in Greece by combined use of Neisseria gonorrhoeae multiantigen sequence typing and multilocus sequence typing.

The prevalence of quinolone-resistant Neisseria gonorrhoeae (QRNG) in Greece remained low from 1997 to 2003 but increased dramatically from 11% to 56% between 2004 and 2007. N. gonorrhoeae multiantigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) were used to investigate trends in quinolone resistance from 1997 to 2007 and explore the origins of the recent increase in QRNG. We characterized 295 QRNG isolates from the study period and 233 quinolone-susceptible (QS) gonococci from 2004 and 2005, when the rapid increase in QRNG occurred. From 1997 to 1999, an outbreak of QRNG was due to the dissemination of isolates of serovar Arst that belonged to two closely related genotypes. Few QRNG isolates, of diverse genotypes, were present between 2001 and 2003, whereas the sharp increase in QRNG from 2004 onwards was due to the appearance of serovar Bropyst isolates of several major NG-MAST sequence type (STs) that previously had not been identified in Greece. These isolates were shown by MLST to be variants of a single multiply antibiotic-resistant QRNG strain (ST1901) that appeared in Greece and rapidly diversified into 31 NG-MAST STs. There were no isolates of MLST ST1901 or any of the 31 NG-MAST STs among QS isolates from 2004 and 2005 or among 8 representatives of multiresistant but quinolone-susceptible serovar Bropyst isolates circulating in Greece during the 1990 s, supporting the view that the recent increase in QRNG was due to importation of a QRNG strain(s) of MLST ST1901 into Greece. Recently, multiresistant QRNG isolates of ST1901 with reduced susceptibility to the newer cephalosporins have appeared in Greece.

Multilocus sequence typing of IncN plasmids.

OBJECTIVES: Incompatibility group N (IncN) plasmids have been associated with the dissemination of antimicrobial resistance and are a major vehicle for the spread of bla(VIM-1) in humans and bla(CTX-M-1) in animals. A plasmid multilocus sequence typing (pMLST) scheme was developed for rapid categorization of IncN plasmids. METHODS: Twelve fully sequenced IncN plasmids available at GenBank were analysed in silico for selecting the loci for the IncN-specific pMLST. A total of 58 plasmids originating from different reservoirs (human, pig, poultry, cattle and horses) and geographic regions (Italy, Greece, Denmark, UK and The Netherlands) were classified by DNA sequencing of the amplicons obtained for the repA, traJ and korA loci. RESULTS: Eleven sequence types (STs) were defined on the basis of allele sequences of the three selected loci. Most plasmids carrying bla(CTX-M-1) (24/27) isolated in different countries from both animals and humans belonged to ST1, suggesting dissemination of an epidemic plasmid through the food chain. Fifteen of 17 plasmids carrying bla(VIM-1) from Klebsiella pneumoniae and Escherichia coli, isolated during a 5 year period in Greece were assigned to ST10, suggesting that spread and persistence of this particular IncN-carrying bla(VIM-1) lineage in Greece. CONCLUSIONS: This study proposes the use of pMLST as a suitable and rapid method for identification of IncN epidemic plasmid lineages. The recent spread of bla(CTX-M-1) among humans and animals seems to be associated with the dissemination of an epidemic IncN plasmid lineage.

An update of the evolving epidemic of blaKPC-2-carrying Klebsiella pneumoniae in Greece (2009-10).

OBJECTIVES: To follow the epidemic of KPC-2-producing Klebsiella pneumoniae in Greece. METHODS: KPC-2-producing isolates (n = 378) were collected during January 2009-April 2010 in 40 Greek hospitals. bla(KPC) and bla(VIM) were detected by PCR. Carbapenemase production was confirmed by spectrophotometry. Sequences flanking bla(KPC-2) and their plasmid carriers were studied. Isolates were typed by PFGE and multilocus sequence typing (MLST). RESULTS: All 378 isolates were bla(KPC-2) positive; 18 also carried bla(VIM-1/VIM-4). Higher isolation frequencies were observed in Athens and Crete. Isolates were classified into 13 PFGE types and 11 sequence types (STs). ST258 was predominant (n = 322), followed by ST147 (n = 20), ST383 (n = 9), ST133 (n = 6), ST274 (n = 4) and ST323 (n = 3). Of the remaining isolates, seven were distributed into five STs (11, 17, 340 and the novel 494 and 495) and seven were not typed. bla(KPC-2) could not be transferred from ST258 isolates, in contrast to isolates of ST17, ST133, ST147, ST274, ST494 and ST495. All bla(KPC-2)-encoding plasmids were of similar size (∼100 kb) and showed indistinguishable restriction fragment length polymorphism (RFLP) patterns except those from the ST340 isolates. Sequences flanking bla(KPC-2) revealed that the Tn4401a isoform was present in plasmids from all STs except ST340 containing Tn4401b. Co-production of VIM enzymes was observed in isolates of ST147, ST323 and ST383. CONCLUSIONS: Apart from the epidemic of KPC-2-producing K. pneumoniae belonging to ST258 in Greece, diffusion of bla(KPC-2) to at least 10 additional STs has taken place. Notably, strains from three of the latter STs (147, 323 and 383) were found to carry both bla(KPC-2) and bla(VIM).

Detection of carbapenemase producing enterobacteria using an ion sensitive field effect transistor sensor.

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.