A Putative ß-lactamase from Inquilinus limosus

PINO MARYLÚ; DI CONZA JOSÉ; GUTKIND GABRIEL

Buenos Aires

Simposio; IMBS Symposium.2; 2009

UBA

Inquilinus limosus is a non-fermenting Gram negative bacillus of the a-Proteobacteria class, family Rhodospirillaceae. This species is increasingly isolated from cystic fibrosis patients, perhaps due to acquisition of a multiple resistance pattern. However, the associated antibiotic resistance mechanisms are still unknown.  The analyzed isolate was obtained from a CFP at Hospital de Niños Dr. O. Alassia, Santa Fe, Argentina, in 2006. Although disk diffusion susceptibility tests for this microorganism are not yet standardized by CLSI, no inhibition zone could be observed around disks containing colistin, gentamicin, trimethoprim-sulfamethoxazole and either ß-lactam antibiotic or ß-lactam/ß-lactamase inhibitor combination. However, inhibition zone greater than 30 mm for carbapenems and amikacin could be seen. Both Nitrocefin and the iodometric method could reveal ß-lactamase activity from the protein extract.  Our goal is to describe the ß-lactamase enzyme/s present in I. limosus.Material and methodsFragments were cloned into the vector pK19 and then transformed into E. coli TOP 10 F-, selecting with kanamycin and ampicillin. Synergy tests were performed using boronic acid disks (Britania). ß-lactamase activity of crude extracts was detected using Nitrocefin and the iodometric method employing ampicillin as substrate. Multiplex PCR-amplification for known  enterobacterial  ampC  genes was attempted both on  DNA of I. limosus and the corresponding clone. DNA sequence of selected clone was analyzed using on-line bioinformatic tools.ResultsOne clone was obtained (INQK41) with an insert of approximately 9 kb.  This clone was resistant to ampicillin and sensitive to cefepime. In addition, synergy with boronic acid was observed. No enterobacterial  ampC genes could  be detected. Crude extracts of INQK41 and I. limosus showed ß-lactamase activity. The enzymatic activity could not be inhibited by clavulanate or sulbactam. Some 6 kb could be sequenced up to date, showing a high GC content (average 70 %).  From these, a 1083 bp region presents homology with several ß-lactamases coding genes deposited in databases. The predicted ORF codified a protein of 361 amino acid with an unusual start codon (GTG). A signal peptide could be predicted. Typical motifs of ß-lactamases in precise position (SXXK element, YSD loop and HTG element) were observed. The active site serine is present at a position common to class A and D ß-lactamases. No omega loop, characteristic of class A enzymes, could be clearly assigned.ConclusionThe cloned fragment of I. limosus chromosome presents a putative ORF that could encode a ß-lactamase enzyme. The activity detected on the crude extracts strongly support this hypothesis. To determine the protein activity, cloning, expression and purification assays are being carried out.