Novel β-Lactamase Genes from Two Environmental Isolates of Vibrio harveyi

Two ampicillin-resistant (Ampr ) isolates ofVibrio harveyi , W3B and HB3, were obtained from the coastal waters of the Indonesian island of Java. Strain W3B was isolated from marine water near a shrimp farm in North Java while HB3 was from pristine seawater in South Java. In this study, novel β-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 for thebla gene of strain W3B while an ORF of 849 bp encoding a 283-amino-acid protein (VHH-1) was deduced forbla 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 homology to any other β-lactamases in the databases. The deduced proteins were found to be class A β-lactamases bearing low levels of homology (<50%) to other β-lactamases of the same class. The highest level of identity was obtained with β-lactamases fromPseudomonas aeruginosa , i.e., PSE-1, PSE-4, and CARB-3, andVibrio cholerae CARB-6. Our study showed that both strains W3B and HB3 possess an endogenous plasmid of approximately 60 kb in size. However, Southern hybridization analysis employingbla VHW-1 as a gene probe demonstrated that thebla gene was not located in the plasmid. A total of nine ampicillin-resistantV. harveyi strains, including W3B and HB3, were examined by pulsed-field gel electrophoresis ofNot I-digested genomic DNA. Despite a high level of intrastrain genetic diversity, thebla VHW-1 probe hybridized only to an 80- or 160-kbNot I genomic fragment in different isolates.