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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.

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

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

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.

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).

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.

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.

An extended-spectrum beta-lactamase, IBC-2, produced by a clinical strain of Pseudomonas aeruginosa, was characterized. bla(IBC-2) was found, as a gene cassette, to be the sole gene within the variable region of a class 1 integron probably located in the chromosome. IBC-2 is a variant of IBC-1 and GES-1, differing by one amino acid from each of these beta-lactamases. When expressed in Escherichia coli, IBC-2 was observed to confer resistance to ceftazidime and decreased susceptibility to other oxyimino-beta-lactams.

GES-13 β-lactamase, a novel GES variant possessing Lys-104 and Asn-170, was identified in Pseudomonas aeruginosa. bla GES-13 was the single gene cassette of a class 1 integron probably located in the chromosome. GES-13 efficiently hydrolyzed broad-spectrum cephalosporins and aztreonam. Imipenem was a potent inhibitor of GES-13 but was not hydrolyzed at measurable rates.

Seven multiresistant Klebsiella pneumoniae strains isolated in an intensive care unit were studied. Susceptibility to beta-lactams was determined with the E test. Molecular-typing of the isolates was performed by a PCR-based technique (ERIC2-PCR). Sonic cell-extracts were used as beta-lactamase preparations. Beta-lactamase quantities were evaluated measuring nitrocefin hydrolysis. The similarity of the ERIC2-PCR patterns indicated that the seven isolates constituted a single clone. The levels of resistance to oximino-beta-lactams, however, were different. There were indications that the differences in susceptibilities were due, at least partly, to differences in the levels of expression of an extended-spectrum beta-lactamase (most likely SHV-5). Related K. pneumoniae isolates may exhibit different levels of resistance to beta-lactams. Therefore, comparison of resistance phenotypes is of limited usefulness in epidemiological investigations of nosocomial infections caused by resistant K. pneumoniae.

VIM-1-producing Klebsiella pneumoniae strains, increasingly isolated in Greece since 2002, constitute the majority of multiresistant clinical isolates of this species found in most Greek hospitals ([9][1]). Moreover, following reports on Klebsiella pneumoniae carbapenemase (KPC)-producing isolates

management of cryptococcal disease. Clin Infect Dis 2000; 30: 710–8. 3. Sable CA, Strohmaier KM, Chodakewitz JA. Advances in antifungal therapy. Annu Rev Med 2008; 59: 455–73. 4. Baddley JW, Pappas PG. Antifungal combination therapy. Drugs 2005; 65: 1461–80. 5. Sionov E, Mendlovic S, Segal E. Efficacy of amphotericin B or amphotericin B-intralipid in combination with caspofungin against experimental aspergillosis. J Infect 2006; 53: 131–9. 6. Mukherjee PK, Sheehan DJ, Hitchcock CA et al. Combination treatment of invasive fungal infections. Clin Microbiol Rev 2005; 18: 163–94.

Infections due to multidrug- and carbapenem-resistant isolates of Acinetobacter baumannii were frequent among patients treated in the Tzannion General Hospital in Piraeus, Greece, throughout the period 2005 to 2006. Six of these isolates, derived from blood cultures of patients in three different wards (four were from patients in the intensive care unit) and taken at least 1 month apart from each other, were studied. Species identification, initially performed with the API 20NE system (bioMerieux, Marcy l'Etoile, France), was confirmed by sequencing of 16S rRNA genes. Antimicrobial MICs were determined by agar dilution. Screening for metallo-β-lactamases (MBLs) was performed with imipenem-EDTA synergy tests: (i) the MBL Etest (AB Biodisk), (ii) the double-disc synergy test (3), and (iii) the combined-disc test (1). β-Lactamases were studied by isoelectric focusing of crude cell extracts derived by sonication. The carbapenemase activity of β-lactamase-containing extracts was assessed by spectrophotometry using imipenem (100 μM) as the reporter substrate and is expressed as units (1 U was the amount of enzyme hydrolyzing 1 μmol of imipenem per min per mg of protein). Reaction mixtures were supplemented with ZnSO4 (50 μM) (6). Imipenem hydrolysis rates were also determined in the presence of EDTA at a final concentration of 100 μM. Preliminary experiments using extracts from Escherichia coli laboratory strains containing various β-lactamases showed that EDTA at 100 μM inhibited VIM-1 activity by >95%, while the activities of OXA, TEM, and AmpC enzymes were virtually unaffected. The blaOXA and MBL genes were identified by PCR as described previously (10) and by sequencing. The association of blaOXA genes with ISAba elements was studied by PCR (11). blaVIM-1-carrying integrons were characterized by the sequencing of overlapping PCR products based on published sequences (7, 8). The isolates were typed by pulsed-field gel electrophoresis (PFGE) (9). The location of the VIM-1-encoding integrons was determined by an I-CeuI-based method (5). The isolates were highly resistant to piperacillin-tazobactam, cefotaxime, ceftazidime, cefepime, and aztreonam (MICs, 64 to >128 μg/ml). Imipenem and meropenem MICs ranged from 16 to 32 μg/ml. The isolates were also resistant to ampicillin-sulbactam, gentamicin, amikacin, tobramycin, and ciprofloxacin. Only one isolate was MBL positive by the double-disc synergy test, though it tested negative by the MBL Etest and the combined-disc test. The remaining five isolates appeared to be negative by all three tests. Notwithstanding the minor phenotypic differences, the isolates exhibited similar PFGE patterns (>80% relatedness), suggesting a clonal relationship. The isolates carried the carbapenemase genes blaOXA-51 and blaOXA-58. ISAba3 was found by PCR upstream of blaOXA-58. Moreover, ISAba1 was adjacent to blaOXA-51, probably providing the promoter for this gene (11). Other types of blaOXA genes were not identified. Unlike with the results of the MBL detection tests, all six isolates contained the blaVIM-1 MBL gene. The results of the isoelectric focusing experiments were in line with the bla gene content. The isolates produced β-lactamases with isoelectric points (pIs) of approximately 6.5 and 7.0, consistent with OXA enzymes, as well as an enzyme with an alkaline pI (∼9.0), probably representing the chromosomal cephalosporinase of this species. A β-lactamase with a pI equal to that of VIM-1 (5.1) that was inhibited in situ by EDTA was also detected, indicating MBL expression. The levels of imipenem hydrolysis by the extracts of five blaVIM-1 carriers were similar, ranging from 28 to 36 U. The remaining blaVIM-1-positive isolate (TZ42/166) exhibited significantly higher activity against imipenem (90 U). These activities were at least 10-fold higher than those of 10 MBL-negative, carbapenem-resistant OXA-58 producers (obtained from two hospitals in Athens, Greece, in 2007) tested in parallel. EDTA reduced imipenem hydrolysis at levels similar to those of the blaVIM-1-negative isolates (1.5 to 3 U), suggesting that carbapenemase activities were largely due to VIM-1. The isolates contained a class 1 integron designated In-Ab10a with a variable region of 1,725 bp comprising, from 5′ to 3′, aacA4 and blaVIM-1. The promoter region included a strong P1 and the inactive form of P2. Similar integrons have been identified in a VIM-1-encoding plasmid from an Escherichia coli isolate also from Greece (8), as well as in VIM-4-producing Pseudomonas aeruginosa isolates from Hungary and Poland (4, 7). The blaVIM cassettes in In-Ab10a and the related integrons contain, between the coding sequence and 59-be, a 170-bp direct repeat of the 3′ end of blaVIM-1. It has been proposed that this unusual structure evolved through a partial deletion of a tandem blaVIM cassette (7). In addition to In-Ab10a, isolate TZ42/166 harbored a novel integron, In-Ab10b, including only the characteristic blaVIM-1 cassette. The presence of two VIM-1-encoding integrons in TZ42/166 may explain the relatively higher carbapenemase activity; however, the higher carbapenemase activity did not seem to significantly affect β-lactam resistance levels. blaVIM-1 was detected by hybridization in an I-CeuI fragment of approximately 250 kb (Fig. ​(Fig.1),1), documenting the chromosomal location of In-Ab10a and In-Ab10b. FIG. 1. (A) Structure of the blaVIM-1-carrying integron In-Ab10a. The direction of transcription is indicated by the arrows. The dotted rectangle to the left of the duplicated 3′ end of blaVIM-1 (Pseudo 59-be) represents the inverse core sequence of the ... The above findings indicate a protracted hospital outbreak caused by genetically related A. baumannii isolates producing VIM-1. A. baumannii isolates are routinely examined by EDTA-imipenem tests in Greek hospitals, where VIM enzymes are widespread. This approach has allowed the detection of sporadic strains carrying In-e541, a VIM-1-encoding integron that, like In-Ab10a, is also spread in enterobacteria (10). Additionally, a PCR-based screening in a hospital in northern Greece of 78 carbapenem-resistant isolates that were negative by imipenem-EDTA synergy tests revealed two blaVIM-1 carriers (2). Taken together, these studies suggest that blaVIM-1-positive A. baumannii strains have been established in this setting and that the conventional EDTA-based tests may not be suitable for their detection. The precise contribution of VIM-1 to the overall β-lactam resistance was not assessed. Nevertheless, the failure of the phenotypic tests to detect VIM-1 in these isolates underscores the role of multiple mechanisms, such as the production of OXA enzymes in determining carbapenem resistance in this species.

This study exploited the possibility to detect Citrobacter freundii -derived CMY-2-like cephalosporinases in Enterobacteriaceae clinical isolates using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Periplasmic proteins were prepared using a modified sucrose method and analyzed by MALDI-TOF MS. A ca. 39,850- m / z peak, confirmed to represent a C. freundii -like β-lactamase by in-gel tryptic digestion followed by MALDI-TOF/TOF MS, was observed only in CMY-producing isolates. We have also shown the potential of the assay to detect ACC- and DHA-like AmpC-type β-lactamases.

CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli emerged recently in Cameroon. CTX-M-15 was encoded by two different multiresistance plasmids, of which one carried an IS Ecp1 - bla CTX-M-15 element flanked by a 5-bp target site duplication and inserted within a Tn 2 -derived sequence. A truncated form of this element in the second plasmid was identified.

The virulence of a KPC-producing Klebsiella pneumoniae sequence type 258 (ST258) strain representing those circulating in Greece was assessed in a mouse septicemia model. The strain was virtually avirulent (50% lethal dose, >10 8 and 5 × 10 7 CFU for immunocompetent and neutropenic animals, respectively). Also, it was highly susceptible to serum killing, rapidly phagocytosed in vitro , and classified as K41, which is not among the virulent capsular types. The findings indirectly support the notion that high ST258-associated mortality is largely due to inefficient antimicrobial treatment.

OBJECTIVES To determine the complete nucleotide sequence of the VIM-1-encoding plasmid pKP1780 from Klebsiella pneumoniae ST147 representing a distinct group of IncR replicons. METHODS The plasmid pKP1780 was from a K. pneumoniae clinical strain (KP-1780) isolated in Greece in 2009. Plasmid DNA was extracted from an Escherichia coli DH5α transformant and sequenced using the 454 Genome Sequencer GS FLX procedure on a standard fragment DNA library. Contig gaps were filled by sequencing of PCR-produced fragments. Annotation and comparative analysis were performed using software available on the Internet. RESULTS Plasmid pKP1780 (49 770 bp) consisted of an IncR-related sequence (12 083 bp) including replication and stability systems, and a multidrug resistance (MDR) mosaic region (37 687 bp). blaVIM-1 along with the aacA7, dfrA1 and aadA1 cassettes comprised the variable region of an integron similar to In-e541 from pNL194. The mosaic structure also included the strA, strB, aphA1 and mphA resistance genes as well as intact (n = 10) or defective (n = 3) insertion sequences and fragments of various transposons. CONCLUSIONS The mosaic structure of pKP1780 exhibited high similarity with the acquired region of the IncN plasmid pNL194, indicating the acquisition of the VIM-1-encoding MDR region from pNL194 by an IncR-type plasmid.

Cefoxitin resistance in Klebsiella pneumoniae from Escherichia coli strains isolated in Greek hospitals was found to be due to the acquisition of similar plasmids coding for group 1 beta-lactamases. The plasmids were not self-transferable but were mobilized by conjugative plasmids. These elements have also been spread to Enterobacter aerogenes. The most common enzyme was a Citrobacter freundii-derived cephalosporinase (LAT-2) which differed from LAT-1 by three amino acids.

The in vitro activity of cefetamet against Escherichia coli, Klebsiella pneumoniae and Serratia marcescens strains expressing an SHV-5-type beta-lactamase was evaluated. Most of the examined strains were susceptible to the antibiotic. Cefetamet was found to be considerably more active than ceftazidime and aztreonam. Its activity was comparable to that of cefotaxime. Cefetamet MIC values were related to the quantity of the enzyme expressed. The SHV-5-type enzyme hydrolysed cefetamet with an efficiency (Vmax/Km) similar to that of ceftazidime.

An IncN plasmid (p541) fromEscherichia colicarried aCitrobacter freundii-derived sequence of 4,252 bp which included anampC-ampRregion and was bound by two directly repeated IS26elements.ampCencoded a novel cephalosporinase (CMY-13) with activity similar to that of CMY-2. AmpR was likely functional as indicated in induction experiments.

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-β-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).

An Escherichia coli strain exhibiting decreased susceptibility to carbapenems was isolated from a hospitalized patient in Greece. The strain carried a self-transferable plasmid coding for metallo-β-lactamase VIM-1. bla VIM-1 , along with aacA7 , dhfrI , and aadA , was included as a gene cassette in a novel class 1 integron. A Citrobacter freundii ampC -derived gene, not associated with the integron, was also located in the same plasmid.

VEB-1-producing Klebsiella pneumoniae strains have recently been identified in Greece ([5][1]). We report here on a VEB-1-positive and multidrug-resistant Proteus mirabilis isolate (PM91) recovered from a patient treated in a Greek hospital in 2010. PM91 was resistant to penicillins, penicillin

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.

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).

VIM-27 metallo-β-lactamase, an Ala 57 → Ser variant of VIM-1, was identified in three Klebsiella pneumoniae isolates belonging to sequence type 147. bla VIM-27 was part of a class 1 integron carried by non-self-transferable plasmids. Kinetic parameters and MIC determinations indicated that VIM-27 hydrolyzed most β-lactams, especially imipenem and cefoxitin, less effectively than VIM-1.

Proteus mirabilis isolates obtained in 1999 to 2008 from three European countries were analyzed; all carried chromosomal AmpC-type cephalosporinase bla CMY genes from a Citrobacter freundii origin ( bla CMY-2 -like genes). Isolates from Poland harbored several bla CMY genes ( bla CMY-4 , bla CMY-12 , bla CMY-14 , bla CMY-15 , and bla CMY-38 and the new gene bla CMY-45 ), while isolates from Italy and Greece harbored bla CMY-16 only. Earlier isolates with bla CMY-4 or bla CMY-12 , recovered in France from Greek and Algerian patients, were also studied. All isolates showed striking similarities. Their bla CMY genes resided within IS Ecp1 transposition modules, named Tn 6093 , characterized by a 110-bp distance between IS Ecp1 and bla CMY , and identical fragments of both C. freundii DNA and a ColE1-type plasmid backbone. Moreover, these modules were inserted into the same chromosomal site, within the pepQ gene. Since ColE1 plasmids carrying IS Ecp1 with similar C. freundii DNA fragments (Tn 6114 ) had been identified earlier, it is likely that a similar molecule had mediated at some stage this DNA transfer between C. freundii and P. mirabilis . In addition, isolates with bla CMY-12 , bla CMY-15 , and bla CMY-38 genes harbored a second bla CMY copy within a shorter IS Ecp1 module (Tn 6113 ), always inserted downstream of the ppiD gene. Sequence analysis of all mobile bla CMY-2 -like genes indicated that those integrated in the P. mirabilis chromosome form a distinct cluster that may have evolved by the stepwise accumulation of mutations. All of these observations, coupled to strain typing data, suggest that the bla CMY genes studied here may have originated from a single IS Ecp1 -mediated mobilization-transfer-integration process, followed by the spread and evolution of a P. mirabilis clone over time and a large geographic area.

The sequence of pR3521, a self-transmissible plasmid from Escherichia coli , was determined. pR3521 (110,416 bp) comprised a contiguous IncB sequence (84,034 bp) sharing extensive similarities with IncI replicons and an acquired region (26,382 bp) carrying sequences of diverse origin, containing bla ACC-4 , bla SCO-1 , bla TEM-1b (two copies), strA , strB , sul2 , and aacC2 .

The nucleotide sequence of pNL194, a VIM-1-encoding plasmid, is described in this study. pNL194 (79,307 bp) comprised an IncN-characteristic segment (38,940 bp) and a mosaic structure (40,367 bp) including bla VIM-1 , aacA7 , aadA1 , aadA2 , dfrA1 , dfrA12 , aphA1 , strA , strB , and sul1 . Tn 1000 or Tn 5501 insertion within fipA probably facilitated recruitment of additional mobile elements carrying resistance genes.

Department of Biology, Indiana University, Bloomington, IN, USA; Lahey Clinic, Burlington, MA, USA; University of L’Aquila, L’Aquila, Italy; Louis Stokes Cleveland Department of Veteran Affairs Medical Center, Cleveland, OH, USA; Infectious Disease Discovery Research, Wyeth Research, Pearl River, NY, USA; Department of Pathology, University of Verona, Italy; Hospital Universitari Mutua de Terrassa, Barcelona, Spain; Athens Medical School, Athens, Greece; Assistance Publique-Hopitaux de Paris, France; Service of Microbiology, University Hospital Marques de Valdecilla, Santander, Spain; Laboratory of Bacteriology, Hellenic Pasteur Institute, Athens, Greece; Department of Pharmacology, Baylor College of Medicine, Houston, TX, USA; School of Medicine, Sevilla, Spain; Hospital Universitario Virgen Macarena, Seville, Spain; Dipartimento di Biologia Molecolare, Universita di Siena, Siena, Italy; Laboratoire de Bacteriologie LRMA, Faculte de Medecine UPMC, Paris, France; Department of Microbiology, National School of Public Health, Athens, Greece

The relative strengths of the uncommon promoters hybrid 2, hybrid 1 with an active P2 promoter (hybrid 1+P2), and strong+P2, which drive transcription of resistance genes in class 1 integrons, were evaluated using bla GES-1 as a reporter gene cassette. Hybrid 2 was stronger than hybrid 1. Coupling P2 with the strong promoter and with hybrid 1 caused a measurable increase in GES-1 expression.

Objectives: To characterize the antibiotic resistance regions of pCC416, a VIM-4- and CMY-4-encoding plasmid from clinical enterobacteria, and to elucidate its relation with the CMY-encoding plasmids widely diffused in Salmonella. Methods: The enterobacterial multiresistant plasmid pCC416 was derived from an Escherichia coli transconjugant and characterized. Conventional and long-range PCR assays were performed using primers specific for VIM-4- and CMY-4-encoding segments of pCC416. Amplicons were characterized by sequencing. blaVIM-4, blaMY-4 and IntI1-specific probes were prepared from PCR products and used for the identification of various pCC416 clones. VIM- and CMY-positive Bam HI and Sau 3AI fragments of pCC416 were cloned into pACYC184 and their sequences were determined by gene walking. Results: The pCC416 plasmid contained two distinct resistant loci carrying β-lactamase genes. The blaVIM-4 gene was part of an integron located in a complex, multidrug-resistant region of novel structure, interspersed with mobile elements or remnants thereof and being similar to various regions of other resistance plasmids. Nevertheless, a region in the 3′ end of this structure resembled the respective region found in a CMY-2-encoding plasmid from Salmonella. The blaCMY-4 gene was identified within an 11.3 kb region also related to the CMY-2-encoding plasmids. Conclusions: pCC416 probably evolved from an IncA/C2, CMY-encoding plasmid through acquisition of a VIM-encoding In4-type integron providing an example of accretion of resistance determinants in a single replicon. © The Author 2007.

The effect of replacement of Met-69 by Ile or Val on the properties of the extended-spectrum β-lactamase SHV-5 was studied. Mutant enzymes were constructed by site-specific mutagenesis and expressed under isogenic conditions in Escherichia coli DH5α cells. Compared with SHV-5, the mutant β-lactamases conferred lower levels of β-lactam resistance and were less efficient in hydrolyzing ampicillin, cephalothin, and cefotaxime. The substitutions rendered SHV-5 less susceptible to inhibition by clavulanate, sulbactam, and tazobactam; however, the MICs of penicillin-inhibitor combinations remained similar, suggesting an attenuation of penicillinase activity.

Objectives: To elucidate the mechanisms responsible for the diversity of b-lactam resistance phenotypes among isolates of a VIM-1-producing Klebsiella pneumoniae (VPKP) strain that is endemic in Greek hospitals. Methods: Five VPKP clinical isolates were studied. MICs of b-lactams were determined by agar dilution. PFGE of XbaI-digested genomic DNA was used for typing. Profiles of outer membrane proteins (OMPs) were determined by SDS–PAGE. Selected isolates were transformed with a plasmid encoding the Omp36K porin. b-Lactamase activities were analysed by IEF and imipenem hydrolysis was assessed by spectrophotometry. VIM-1-encoding, self-transmissible plasmids were characterized by replicon typing, RFLP and hybridization with blaVIM- and IS26-specific probes. Characterization of integrons was performed by PCR, cloning and sequencing. Results: Isolates exhibited highly similar PFGE patterns. Imipenem MICs were 2, 4, 16, 32 and 64 mg/L. The isolate with the highest imipenem MIC (Vipm-64) lacked a 36 kDa OMP. Expression of a cloned OmpK36 in this isolate reduced the imipenem MIC to susceptibility levels. Imipenem-hydrolysing activity was significantly higher in Vipm-16 as compared with the other isolates that expressed similar amounts of VIM-1. All isolates transferred b-lactam resistance to Escherichia coli through conjugative, IncN plasmids that exhibited differences in the RFLP and hybridization patterns with blaVIM- and IS26-specific probes. The Vipm-16 plasmid, mediating the higher imipenem MICs among transconjugants, carried two copies of blaVIM-1. Cloning and sequencing showed In-e541-like integrons truncated at the 5 0 CS by insertion of IS26 elements at two different positions. Conclusions: A VIM-1-producing strain of K. pneumoniae has evolved through OMP alterations and rearrangements in the blaVIM-1-carrying plasmid probably mediated by IS26, generating isolates with imipenem MICs ranging from susceptibility to resistance.

An inhibitor-resistant beta-lactamase (SHV-10), derived from an SHV-5 variant (SHV-9), was found in an Escherichia coli clinical isolate. In SHV-10, Ser-130 was replaced by Gly. The enzyme partially retained its ability to hydrolyze penicillins, but its activity against cephalosporins was drastically reduced. A Ser-130-->Gly mutant of the prototype SHV-5, obtained by site-directed mutagenesis, had properties similar to those of SHV-10.

USA. 6. Lefort, A., Lafaurie, M., Massias, L. et al. (2003). Activity and diffusion of tigecycline (GAR-936) in experimental enterococcal endocarditis. Antimicrobial Agents and Chemotherapy 47, 216–22. 7. Muralidharan, G., Getsy, J., Mayer, P., et al. (1999). Pharmacokinetics (PK), safety and tolerability of GAR-936, a novel glycylcycline antibiotic, in healthy subjects. In Program and Abstracts of the Thirtyninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract 416, p. 303. American Society for Microbiology, Washington, DC, USA.

Three distinct multiresistant loci from enterobacterial plasmids each comprised an integron and an IS 26 -associated sequence. Sequence comparison suggested a common ancestral structure that derived from an IS 26 insertion into the 5′ conserved segment of an In4-type integron and evolved through acquisition of gene cassettes and IS 26 -mediated recruitment of additional resistance genes of diverse origin.

fluid, two from surgical drainage sites and one from urine. Identification to species level and determination of MICs of several antibiotics were performed using the PASCO ID/MIC system for Gram-positive organisms (Difco Laboratories, Detroit, MI, USA). The MICs of vancomycin and teicoplanin were also determined by Etest. The MICs of vancomycin and teicoplanin were � 256 and 128 mg/L, respectively. The phenotype of all E. faecium isolates was VanA. The results of the susceptibility testing are listed in the Table. Molecular strain typing was performed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction (PCR) analysis. Genomic DNA of the above nine strains was isolated and cleaved by the restriction endonuclease SmaI. The DNA fragments were detected in 1% pulsed-field certified agarose (Bio-Rad Laboratories, Hercules, CA, USA) with a CHEF-DRIII system (BioRad). The voltage gradient was 6 V/cm. For the first 15 h, the pulse was 1‐10 s and for the remaining 8 h it was 10‐30 s. PFGE showed similar banding patterns (data not shown), so the VRE isolates were assumed to be related strains. The presence of van genes was detected by PCR, as proposed by Dutka-Malen et al. 3 Primers VAN A1 (5� -GCTATTCAGCTGTACTC-3� ) and VAN A2 (5� CAGCGGCCATCATACGG-3� ) were used to amplify a 783 bp fragment of the vanA gene. A 1 kb DNA ladder (Gibco-BRL, Paisley, UK) was used as a size standard. The vanA gene was identified in all tested isolates (data not shown). In the USA, VRE strains reportedly accounted for � 25% of all enterococci isolated in intensive care units, 4

Sir, Resistance to extended-spectrum cephalosporins has emerged among non-typhoid salmonellae (NTS) worldwide due to acquisition of plasmid-mediated b-lactamases including CMY-2. The isolation frequency of CMY-2-producing NTS in the USA has gradually increased since 1996 reflecting the widespread transmission of CMY-2-positive NTS among food animal reservoirs. The blaCMY-2 gene has been disseminated by plasmids to various Salmonella serotypes including Typhimurium. – 4 We report here on a human isolate of Salmonella serotype Typhimurium harbouring a CMY-2-encoding plasmid as well as a chromosomally located copy of blaCMY-2. Salmonella serovar Typhimurium AM19083 was submitted to the National Antimicrobial Resistance Monitoring System in 2003 from Massachusetts. Antimicrobial susceptibility was assessed by broth microdilution (Sensititre, Westlake, OH, USA). The isolate met the phenotypic criteria indicative of class C cephalosporinase production (i.e. resistance or decreased susceptibility to cefoxitin, ceftiofur and ceftriaxone). It was also resistant to ampicillin, amoxicillin/clavulanate and cefalotin, but susceptible to non-b-lactam drugs including streptomycin, sulphonamides, chloramphenicol, tetracycline, nalidixic acid and ciprofloxacin. AM19083 was positive in PCR assays with blaCMY-specific primers, and the sequence of the amplicon (369 bp) was homologous to an internal fragment of blaCMY-2 (from nt 271 to nt 639, in GenBank accession no. X91840). Isoelectric focusing of sonicated cell extracts in polyacrylamide gels containing ampholines showed production of a b-lactamase species with an isoelectric point of .8.4 corresponding to CMY-2. b-Lactam resistance was readily transferred by conjugation in broth cultures to a susceptible Escherichia coli host. The plasmid content analysis indicated transfer of an ~90 kb plasmid (designated p19083) that was positive in hybridization with a blaCMY-2 probe prepared as described previously. 5 Plasmid p19083 belonged to the incompatibility Group I1 (IncI1), as found by a PCR-based Inc/rep typing method. Furthermore, the PstI restriction profile of p19083 resembled restriction type B of the IncI1, CMY-2-encoding plasmids previously described in Salmonella Typhimurium and Salmonella Thompson isolates from the USA. A plasmid DNA preparation from AM19083 was electrophoresed through agarose gels (0.8%). DNA was then transferred to Hybond-N nylon membranes (Amersham Biosciences, Buckingham, UK) and hybridized with a digoxigenin-labelled blaCMY-2 probe. 5 These experiments showed not only the expected hybridization with plasmid p19083, but also a strong signal with the chromosomal material. This was not observed with preparations from two control strains of serotype Typhimurium, one of which (AM18447) also carried two CMY-2-encoding plasmids, p18447-S and p18447-L (~140 and 300 kb, respectively). To clarify this issue, an I-CeuI genomic analysis of isolates AM19083 and AM18447 was performed. The digested fragments were separated by PFGE (6.0 V/cm for 8 h/16 h with a pulsing time linearly ramped from 5 to 20 s/20 to 70 s) (Figure 1a). The blaCMY-2 probe was hybridized with a 16SrRNA-positive I-CeuI fragment of ~500 kb from isolate AM19083. Positive signals probably corresponding to the blaCMY-2-carrying plasmids were also seen (Figure 1b). Subsequent hybridization with Inc/rep probes showed that p19083 and p18447-S belonged to IncI1, whereas the blaCMY-2-carrying putative plasmid p18447-L and the 500 kb fragment from AM 19083 were not reactive (Figure 1c). In an attempt to rule out the existence of blaCMY-2-positive plasmids of high molecular weight in AM19083 and, also, to corroborate the plasmidic nature of p18447-L, the plasmid contents of AM19083 and AM18447 were analysed by PFGE after treatment of total DNA with S1 nuclease. The respective electrophoretic profiles indicated the absence of blaCMY-2-carrying ‘megaplasmids’ in AM19083, whereas a band of ~300 kb likely representing the linear form of p18447-L was observed in the preparations from AM18447 (data not shown). Finally, to further strengthen our hypothesis for the chromosomal acquisition of blaCMY-2 in AM19083, the relevant I-CeuI fragment was purified by electroelution. The resulting preparation contained blaCMY-2 as confirmed by PCR and sequencing. It was also negative in PCR assays designed for replicon typing of plasmids including IncI1 and IncA/C. Nevertheless, the possibility that plasmid segments including rep could have been removed during I-CeuI treatment cannot be excluded. Production of CMY-2 has been described in a wide variety of NTS serotypes in the USA. The plasmidic location of blaCMY-2 is critical for spread of this resistant determinant. The present findings confirm the prominent role of IncI1 plasmids in this process. Additionally, the likely chromosomal carriage of blaCMY-2 underscores the operation of efficient mobilization mechanisms commonly involving ISEcp1 and/or IS26. Multiple copies of a resistance gene may increase the antimicrobial resistance levels and provide more of a selective advantage. Expression of the chromosomal CMY-2 and its possible contribution to b-lactam resistance levels were not studied. b-Lactam MICs for Salmonella Typhimurium AM19083, however, were comparable with those observed for other Salmonella strains carrying only plasmidic blaCMY-2, such as AM18447, but, for many of Journal of Antimicrobial Chemotherapy (2008) 61, 1389–1399 doi:10.1093/jac/dkn116 Advance Access publication 18 March 2008