Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1 I35T and NDM-1 I35S enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. The k cat , K m , and k cat /K m values calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, the k cat /K m of NDM-1 I35S for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1 I35T and NDM-1 I35S enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of ␣-helix content in the mutants. The NDM-1 metallo-␤-lactamase (MBL) was first described in a urinary Klebsiella pneumoniae isolate recovered from a Swedish patient who traveled to New Delhi and who had received medical care in India (1). This is the most recent MBL to have widely spread around the world among enterobacterial strains, Pseudomonas aeruginosa (2), Acinetobacter baumannii (3), Morganella morganii (4), Alcaligenes faecalis, Vibrio cholerae, and Stenotrophomonas maltophilia (5, 6). NDM-1-producing bacteria have been recovered from many infection sites as hospital-acquired and community-acquired infections but also in environmental samples (7). Since its finding, 12 NDM variants have been identified (http://www.lahey.org/Studies/). The factor that has influenced the wide geographic spread of bla NDM-1 gene is its localization on complex plasmids that mediate the transfer of this resistant determinant under the selective pressure of antibiotic therapy (8, 9). Metallo-␤-lactamases show a broad-spectrum substrate profile; they are resistant to classical ␤-lactamase inhibitors and hydrolyze carbapenems very efficiently. MBLs require one or two zinc ions to catalyze the hydrolysis of ␤-lactams. It is commonly suggested that the zinc ion acts as a Lewis acid to stabilize the transient tetrahedral intermediate formed by the nucleophilic attachment of a hydroxide ion to the carboxyl group of the ␤-lactam ring (10). NDM-1 enzyme shows a great ability to hydrolyze all ␤-lactam antibiotics (11). Several crystal structures of NDM-1 have been solved, and the enzyme displays the typical ␣␤/␤␣ fold of MBLs (12,13,14). NDM-1 belongs to subclass B1, and it contains a binuclear Zn active site surrounded by several loops responsible for substrate binding and specificity (13). Several studies pointed out the attention focused on the L3 loop, which could be involved in the hydrophobic contacts with substrates through the presence of an aromatic residue. Moreover, its great flexibility makes the loop able to drive substrate into the active site. The role of the L3 loop has been studied in such subclass B1 MBLs as IMP-1 and VIM-2 (15), but no experimental data are available for the NDM-1 enzyme. In comparison to the IMP and VIM variants, NDM-1 has a longer N terminus which forms two extra strands that pack on the L3 loop through an isoleucin...

A multi-drug resistant strain of E. miricola was isolated from the urine of a 2-year old boy hospitalized for severe clinical conditions. The strain produces two metallo-β-lactamases belonging to subclasses B1 and B3: a new BlaB variant (BlaB-15) and a GOB-7-like enzyme.Dear Editor, I send you a copy of revised manuscript entitled "BlaB-15, a new BlaB metallo-β-lactamase variant found in an Elizabethkingia miricola clinical isolate" by Colapietro M et al. that I would like to submit on Diagnostic Microbiology and Infectious Disease.I state that this manuscript has not been and will not be submitted simultaneous elsewhere for publication.All the authors agree with the instructions and conditions of the Journal.All the authors agree to the submitted draft of the paper.Thank you in advance for your cooperation. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 AbstractA multi-drug resistant strain of E. miricola was isolated from the urine of a 2-year old boy hospitalized for severe clinical conditions. The strain produces two metallo-β-lactamases belonging to subclasses B1 and B3: a new BlaB variant (BlaB-15) and a GOB-7-like enzyme. Urine was collected from an intermittent urinary catheter and showed leucocyte count of 500 cells/μl and presence of nitrites. In addition, the urine sample gave the following results: 10.000 CFU/ml of Klebsiella pneumoniae (ESBL-positive), 10.000 CFU/ml of E. miricola, 100.000 CFU/ml of Pseudomonas aeruginosa, 100 CFU/ml of Candida and 100 CFU/ml of viridans streptococci. The young patient was under long-term treatment with nitrofurantoin. Genomic DNA of E. miricola SW-2 was extracted according to standard procedure (Sambrook et al., 1989). The presence of large plasmid was also ascertained using Kado method , but no plasmid bands were observed in E. miricola SW2. PCR screening was performed using specific primers to identify bla KPC , bla VIM , bla IMP , bla NDM-1 , bla TEM , bla SHV , bla CTX-M determinants as previously reported ; the presence of blaB and bla GOB genes was 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5 open reading frame of 249 amino acids, had more than 90% amino acid identity with BlaB variant.However, some changes in the amino acid sequence make the present BlaB enzyme as new variant, -6, -12, -13) or isoleucine (GOB-8, -9, -10, -18) residues . However, the blaGOB-like gene found in E. miricola was not cloned and GOB-like enzyme was not characterized because it lacks in C-terminal region.The blaB-15 gene was cloned into pET-24(a) vector using NdeI and BamHI restriction sites to obtain the recombinant plasmid pET-BlaB-15 that was inserted by transform...

bSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-␤-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

Two new natural CphA metallo-␤-lactamases, the CphA4 and CphA5 enzymes, were identified in water samples from municipal sewage in central Italy. Compared to CphA, the CphA4 and CphA5 enzymes showed numerous point mutations. These enzymes have a narrow spectrum of substrates focused on carbapenems only. CphA5 showed k cat values about 40-, 12-, and 97-fold higher than those observed for CphA4 versus imipenem, ertapenem, and biapenem, respectively.

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