Novel beta-lactamase genes from two environmental isolates of Vibrio harveyi

Two ampicillin-resistant (Amp(r)) isolates of Vibrio harveyi, W3B and HB3, were obtained from the coastal waters of the Indonesian island of Java. Str ain W3B was isolated from marine water near a shrimp farm in North Java whi le HB3 was from pristine seawater in South Java. In this study, novel beta- lactamase genes from W3B (bla(VHW-1)) and HB3 (bla(VHH-1)) were cloned and their nucleotide sequences were determined. An open reading frame (ORF) of 870 bp encoding a deduced protein of 290 amino acids (VHW-1) was revealed f or the bla gene of strain W3B while an ORF of 849 bp encoding a 283-amino-a cid protein (VHH-1) was deduced for bla(VHH-1). At the DNA level, genes for VHW-1 and VHH-1 have a 97% homology, while at the protein level they have a 91% homology of amino acid sequences. Neither gene sequence showed homolo gy to any other beta-lactamases in the databases. The deduced proteins were found to be class A beta-lactamases bearing low levels of homology (<50%) to other beta-lactamases of the same class. The highest level of identity w as obtained,vith beta-lactamases from Pseudomonas aeruginosa, i.e., PSE-1, PSE-4, and CARB-3, and Vibrio cholerae CARB-6. Our study showed that both s trains W3B and HB3 possess an endogenous plasmid of approximately 60 kb in size. However, Southern hybridization analysis employing bla(VHW-1) as a ge ne probe demonstrated that the bla gene was not located in the plasmid. A t otal of nine ampicillin-resistant V. harveyi strains, including W3B and HB3 , were examined by pulsed-field gel electrophoresis of NotI-digested genomi c DNA. Despite a high level of intrastrain genetic diversity, the bla(VHW-1 ) probe hybridized only to an 80- or 160-kb NotI genomic fragment in differ ent isolates.