Identification, synthesis and biological activity of alkyl-guanidine oligomers as potent antibacterial agents.

In the last two decades, the repertoire of clinically effective antibacterials is shrinking due to the rapidly increasing of multi-drug-resistant pathogenic bacteria. New chemical classes with innovative mode of action are required to prevent a return to the pre-antibiotic era. We have recently reported the identification of a series of linear guanidine derivatives and their antibacterial properties. A batch of a promising candidate for optimization studies (compound 1) turned out to be a mixture containing two unknown species with a better biological activity than the pure compound. This serendipitous discovery led us to investigate the chemical nature of the unknown components of the mixture. Through MS analysis coupled with design and synthesis we found that the components were spontaneously generated oligomers of the original compound. Preliminary biological evaluations eventually confirmed the broad-spectrum antibacterial activity of this new family of molecules. Interestingly the symmetric dimeric derivative (2) exhibited the best profile and it was selected as lead compound for further studies.

Chryseobacterium gleum in a man with prostatectomy in Senegal: a case report and review of the literature.

BACKGROUND: Here we report a rare case of a urinary tract infection due to Chryseobacterium gleum. This widely distributed Gram-negative bacillus is an uncommon human pathogen and is typically associated with health care settings. CASE PRESENTATION: We describe a case of urinary tract infection caused by Chryseobacterium gleum in a 68-year-old man of Wolof ethnicity (an ethnic group in Senegal, West Africa) who presented to our Department of Urology in a university teaching hospital (Hôpital Aristide Le Dantec) in Dakar, Senegal, 1 month after prostatectomy. The strain isolated from a urine sample was identified as Chryseobacterium gleum by mass spectrometry (Vitek matrix-assisted laser desorption/ionization, time-of-flight, bioMérieux) and confirmed by 16S ribosomal ribonucleic acid sequencing. The organism was resistant to a wide range of antibiotics, including carbapenem, due to a resident metallo-ß-lactamase gene that shared 99% of amino-acid identity with Chryseobacterium gleum class B enzym. CONCLUSIONS: Infection by Chryseobacterium gleum is infrequent, and no such case has been previously reported in Africa. Despite its low virulence, Chryseobacterium gleum should be considered a potential opportunistic and emerging pathogen. Further studies on the epidemiology, pathogenicity, and resistance mechanisms of Chryseobacterium gleum are needed for better diagnosis and management.

The three-dimensional structure of VIM-2, a Zn-beta-lactamase from Pseudomonas aeruginosa in its reduced and oxidised form.

The crystal structures of the universally widespread metallo-beta-lactamase (MBL) Verona integron-encoded MBL (VIM)-2 from Pseudomonas aeruginosa have been solved in their native form as well as in an unexpected oxidised form. This carbapenem-hydrolysing enzyme belongs to the so-called B1 subfamily of MBLs and shares the folding of alpha beta/beta alpha sandwich, consisting of a core of beta-sheet surrounded by alpha-helices. Surprisingly, it showed a high tendency to be strongly oxidised at the catalytic cysteine located in the Cys site, Cys221, which, in the oxidised structure, becomes a cysteinesulfonic residue. Its native structure was obtained only in the presence of Tris(2-carboxyethyl)phosphine. This oxidation might be a consequence of a lower affinity for the second Zn located in the Cys site that would also explain the observed susceptibility of VIM-2 to chelating agents. This modification, if present in nature, might play a role in catalytic down-regulation. Comparison between native and oxidised VIM-2 and a predicted model of VIM-1 (which shows one residue different in the Cys site compared with VIM-2) is performed to explain the different activities and antibiotic specificities.

[Detection of metallo-beta-lactamases in gram-negative bacilli isolated in hospitals from Romania--research fellowship report]. / Depistarea producerii de metalo-beta-lactamaze la bacili gram-negativi izolati în spitale din România.

The emergence of carbapenem resistance in Gram-negative bacilli has become a global concern since these beta-lactams are often the only effective treatment left against many multiresistant strains. Metallo-beta-lactamase-producing isolates are now widespread worldwide, and especially in Europe where they have been responsible for rather large outbreaks. Our study proposes to investigate the presence of such isolates in Romania and to characterize them at the molecular level. A total of 28 clinical isolates clinical isolates of Pseudomonas aeruginosa and Acinetobacter baumannii were collected between 2003 and 2007 from 5 hospitals in 3 cities in north-east of Romania (Iasi, Bacau, Galati). Antimicrobial susceptibility testing was performed according to CLSI guidelines. MBL detection was performed using the EPI phenotypic test and PCR, that was confirmed by hydrolysis test. The structure of MBL-encoding class 1 integron was determined by PCR mapping and sequencing. Genotyping was performed by RAPD. Only 2 strains showed a positive EPI test. PCR and sequencing revealed the presence of bla(VIM-2) and bla(IMP-13) genes, in strains 16GL Pa and 247Pa, respectively. RAPD showed clonal relatedness between the Pa247 and an Italian IMP-13 producing strain. Despite the infrequent occurrence of metallo-beta-lactamases in our settings, both VIM- and IMP-types are present in Romanian Pseudomonas aeruginosa clinical isolates.

CENTA as a chromogenic substrate for studying beta-lactamases.

CENTA, a chromogenic cephalosporin, is readily hydrolyzed by beta-lactamases of all classes except for the Aeromonas hydrophila metalloenzyme. Although it cannot practically be used for the detection of beta-lactamase-producing strains on agar plates, it should be quite useful for kinetic studies and the detection of the enzymes in crude extracts and chromatographic fractions.

Molecular heterogeneity of bla(VIM-2)-containing integrons from Pseudomonas aeruginosa plasmids encoding the VIM-2 metallo-beta-lactamase.

A bla(VIM-2) metallo-beta-lactamase determinant, identical to that previously identified in Pseudomonas aeruginosa COL-1 isolate from a French hospital, was detected on a 28-kb plasmid carried by a nosocomial isolate of P. aeruginosa from Verona, Italy. In this plasmid the bla(VIM-2) determinant was inserted into a class 1 integron of original structure, named In72, that contains a partially deleted intI1 integrase gene and two gene cassettes. The first cassette carries an aacA4 aminoglycoside acetyl transferase determinant. The second cassette carries a bla(VIM-2) determinant followed by a partially deleted attC site. The structure of In72 was notably different from that of In56, the bla(VIM-2)-containing integron found in the COL-1 isolate, revealing the existence of molecular heterogeneity among bla(VIM-2)-containing integrons in clinical isolates of P. aeruginosa from Europe.

Metallo-beta-lactamase producers in environmental microbiota: new molecular class B enzyme in Janthinobacterium lividum.

Eleven environmental samples from different sources were screened for the presence of metallo-beta-lactamase-producing bacteria by using a selective enrichment medium containing a carbapenem antibiotic and subsequently testing each isolate for production of EDTA-inhibitable carbapenemase activity. A total of 15 metallo-beta-lactamase-producing isolates, including 10 Stenotrophomonas maltophilia isolates, 3 Chryseobacterium spp., one Aeromonas hydrophila isolate, and one Janthinobacterium lividum isolate (a species in which production of metallo-beta-lactamase activity was not previously reported), were obtained from 8 samples. In the J. lividum isolate, named JAC1, production of metallo-beta-lactamase activity was elicited upon exposure to beta-lactams. Screening of a JAC1 genomic library for clones showing a reduced imipenem susceptibility led to the isolation of a metallo-beta-lactamase determinant encoding a new member (named THIN-B) of the highly divergent subclass B3 lineage of metallo-beta-lactamases. THIN-B is most closely related (35.6% identical residues) to the L1 enzyme of S. maltophilia and more distantly related to the FEZ-1 enzyme of Legionella gormanii (27.8% identity) and to the GOB-1 enzyme of Chryseobacterium meningosepticum (24.2% identity). Sequences related to bla(THIN-B), and inducible production of metallo-beta-lactamase activity, were also detected in the J. lividum type strain DSM1522. Expression of the bla(THIN-B) gene in Escherichia coli resulted in decreased susceptibility to several beta-lactams, including penicillins, cephalosporins (including cephamycins and oxyimino cephalosporins), and carbapenems, revealing a broad substrate specificity of the enzyme. The results of this study indicated that metallo-beta-lactamase-producing bacteria are widespread in the environment and identified a new molecular class B enzyme in the environmental species J. lividum.

Purification and biochemical characterization of the VIM-1 metallo-beta-lactamase.

VIM-1 is a new group 3 metallo-beta-lactamase recently detected in carbapenem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Mediterranean area. In this work, VIM-1 was purified from an Escherichia coli strain carrying the cloned bla(VIM-1) gene by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme exhibited a molecular mass of 26 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an acidic pI of 5.1 in analytical isoelectric focusing. Amino-terminal sequencing showed that mature VIM-1 results from the removal of a 26-amino-acid signal peptide from the precursor. VIM-1 hydrolyzes a broad array of beta-lactam compounds, including penicillins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanism-based serine-beta-lactamase inactivators. Only monobactams escape hydrolysis. The highest catalytic constant/K(m) ratios (>10(6) M(-1). s(-1)) were observed with carbenicillin, azlocillin, some cephalosporins (cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome), imipenem, and biapenem. Kinetic parameters showed remarkable variability with different beta-lactams and also within the various penam, cephem, and carbapenem compounds, resulting in no clear preference of the enzyme for any of these beta-lactam subfamilies. Significant differences were observed with some substrates between the kinetic parameters of VIM-1 and those of other metallo-beta-lactamases. Inactivation assays carried out with various chelating agents (EDTA, 1,10-o-phenanthroline, and pyridine-2,6-dicarboxylic acid) indicated that formation of a ternary enzyme-metal-chelator complex precedes metal removal from the zinc center of the protein and revealed notable differences in the inactivation parameters of VIM-1 with different agents.