A Novel Chromosomal Class C beta-Lactamase in Inquilinus limosus.

PINO MARYLÚ; DI CONZA JOSÉ; GUTKIND GABRIEL

Denver, Colorado

Conferencia; 53rd ICAAC- Annual Interscience Conference on Antimicrobial Agents and Chemotherapy; 2013

American Society of Microbiology

Background: Inquilinus limosus displays a multiresistance pattern, including most beta-lactams antibiotics except carbapenems. We previously reported a chromosomal beta-lactamase INQ-1, with poor catalytic efficiencies which could not explain by itself the observed resistance. Up to date other mechanisms involved in beta-lactams resistance are still unknown. Our goal was to study a putative beta-lactamase presents in the draft genome of I. limosus MP07. Methods: I. limosus MP07 (with high level resistance to beta-lactams) was isolated from an Argentinean CF patient in 2006. The genome sequence was obtained by pyrosequencing and analyzed by on-line bioinformatics tools. The beta-lactamase gene was amplified by PCR and cloned in pET-28b(+) vector. Preliminary kinetic properties were determined spectrophotometrically using an overproduced total protein extract. For poor substrates, apparent Km was determined as Ki using 100μM Nitrocefin as reporter. For inactivators, IC50 was calculated after competition experiments, with and without pre-incubation, using the same reporter. Results: A chromosomal region of 1191 bp showed homology with several beta-lactamase coding genes. By in silico analysis it was predicted an ORF that codify a mature 367- aminoacid putative beta-lactamase (theoretical pI 5.7 and MW 39.4 kDa). The translated sequence displays 67% identity to AmpC of Sinorhizobium fredii and 60% identity to the class C Pseudomonas aeruginosa PDC-13 beta-lactamase. Typical class C beta-lactamase motifs were observed in precise position: SXXK element, YSN loop, KTG element and D217. Neither ampR nor other regulator mechanisms were detected in neighbor genes. The protein extract showed beta-lactamase activity that could be not inhibited by clavulanic acid (Indirect IC50 higer 200 μM). However it could be partially inhibited to sulbactam. On the other hand, 3-phenyl boronic acid exhibited enzymatic inhibition in indirect competitive assays only at high concentrations (Indirect IC50= 50 μM). Apparent Km values for ampicillin, ceftazidime, cefotaxime and cefoxitin were 0.6, 1.5, 0.01 and 0.004 μM, respectively. Conclusion: Kinetic properties of this class C beta-lactamase, named INQ-2, are compatible with others class C beta-lactamase; unusual susceptibility to sulbactam have been reported for other beta-lactamases.