Occurrence and diversity of integrons and beta-lactamase genes among ampicillin-resistant isolates from estuarine waters

Abstract

The aim of the present study was to assess the occurrence and molecular diversity of β -lactamase genes and integrons among Gram-negative ampicillin-resistant bacteria from Ria de Aveiro. Ampicillin-resistant isolates were selected and subjected to genotyping using REP-PCR. Rep- resentatives from each REP pattern were affiliated with the following taxa by sequencing the 16S rRNA gene: Aeromonas caviae, A. hydrophila , A. media, A. molluscorum, A. veronii, A. salmonicida, Aeromonas sp., Pseudomonas putida, Pseudomonas sp., Escherichia coli , Escherichia sp., Shigella sonnei , Shigella sp., Klebsiella pneumoniae , K. oxytoca, Raoultella ornithinolytica , R. terrigena, R. planticola , Citrobacter fre- undii , Morganella morganii and Enterobacter sp. Isolates affiliated with genera Escherichia or Shigella were identified as Escherichia coli using phenotypic-based tests. PCR was used to assess β -lactamase encoding sequences ( bla TEM , bla SHV , bla CARB , bla CTX-M , bla IMP , bla VIM , bla CphA/IMIS , bla OXA-A , bla OXA-B , bla OXA-C ), class 1 and class 2 integrases, and integron variable regions. Sequence analysis of PCR products was performed. β -Lactamase genes were detected in 77.8% of the Enterobacteriaceae and in 10.5% of the Aeromonas . The most frequently detected gene was bla TEM , followed by bla SHV, bla OXA-B , bla CphA/IMIS and bla CARB . Retrieved sequences shared high homology with pre- viously described β -lactamases. The intI1 gene was present in 29.6% of the Enterobacteriaceae and in 21% of the Aeromonas isolates. The intI2 gene was present in 4 isolates. A total of 13 cassettes included in 12 different cassette arrays were identified. The most frequently found resistance gene cassettes were aadA variants. Previous investigations based on cultivation-independent approaches revealed higher molecular diversity among β -lactamase-encoding sequences in this estuary. This fact reinforces the hypothesis that cultivation-dependent approaches may underestimate the prevalence of antibiotic resistance genes in environmental samples and may introduce bias in the recovery of their molecular variants.