Identification of an Inquilinus limosus ß-Lactamase.

PINO MARYLÚ; GABRIEL GUTKIND; DI CONZA JOSÉ

Boston

Congreso; 50th ICAAC Interscience Conference on Antimicrobial Agents and Chemotherapy; 2010

American Society for Microbiology

BackgroundInquilinus limosus are non-fermenting Gram negative bacilli of the a-Proteobacteria class. This species is increasingly isolated from cystic fibrosis patients (CFP), and in general, it shows a multiple resistance pattern. However, the associated antibiotic resistance mechanisms are still unknown. Our goal is to describe the ß-lactamase/s present in I. limosus.Material and methodsI. limosus was isolated from a CFP in a pediatric hospital. Screening and susceptibility tests were performed according CLSI recommendations even in the absence of interpretative breakpoints. Genomic fragments were cloned into pK19 and transformed into E. coli TOP 10 F-. ß-lactamase activity was detected spectrophotometrically using nitrocefin.Multiplex PCR for known enterobacterial ampC genes was attempted. DNA sequence was analyzed using on-line bioinformatic tools.ResultsNo inhibition zone could be observed around any ß-lactam antibiotic or ß-lactam/ß-lactamase inhibitor disks except for carbapenems with the clinical isolate. A clone containing a 9 kb-chromosomal fragment was selected with ampicillin, but remained susceptible to cefepime and intermediate for cefoxitin; synergy with boronic acid was observed. No enterobacterial ampC genes could be detected.The 9 kb-fragment showed a high GC content (70 %). From these, a 1083 bp region presents homology with several ß-lactamase coding genes. The predicted ORF codified a 361-aa protein with an unusual start codon (GTG). Typical classes A/C ß-lactamases motifs in precise position (SXXK element, YSD loop and HTG element) were observed. However, no clear omega-loop could be assigned.Crude extracts from the original isolate and the clone displayed β-lactamase activity that could only be partially inhibited clavulanic acid or 3-aminophenylboronic acid, even after 30 min preincubation.ConclusionThe I. limosus fragment presents a putative ORF that could encode a ß-lactamase. The activity detected on the crude extracts strongly support this hypothesis. However, other mechanisms may be present in the original isolate for explaining full resistance to ß-lactams, as it is common in other gram negative bacilli.