| The psychrotrophic Gram-negative bacteria are the single major cause of organoleptic spoilage of meats at refrigeration temperatures. However, the possible role of these microorganisms in antibiotic resistance is not well understood. In this study, the plasmids obtained from five members of this bacterial group, identified as species of the genera Pseudomonas, Psychrobacter, Acinetobacter, Alcaligenes, and Edwardsiella, were characterized as putative reservoirs for antibiotic-resistance genes. DNA-hybridization analysis using digoxigenin-labeled probes revealed significant relatedness among these cryptic plasmids derived from both psychrotrophic and mesophilic-reference strains. Comparative agarose gel electrophoresis analyses of plasmid DNA using the lambda HindIII fragments revealed that the plasmid sizes ranged from ≤2.0- to ≥23.1-kb, while linear-regression analysis with the supercoiled DNA ladder demonstrated plasmid sizes ranging from 1.540- to 34.261-kb. Plasmid restriction-fragment analyses generated a spectrum of fragments with a significant number of similar-sized fragments shared among these strains. Plasmid-mediated beta-lactamase production was detected and confirmed by polymerase chain reaction (PCR ) using synthetic-oligonucleotide primers, targeting Pseudomonas-specific enzyme (pse-2) and TEM-76 beta-lactamase genes. Amplified products of 600- and 526-by were obtained using the pse -2 and TEM-76 beta-lactamase-derived primers, respectively. TEM beta-lactamase was inducible. Plasmid-cured strains became susceptible to nalidixic acid, trimethoprim/sulfamethoxazole, erythromycin, and carbenicillin. Plasmid loss also affected the expression of several metabolic enzymes, including alkaline and acid phosphatases, valine-, cystine-, and leucine-aminopeptidases, trypsin, esterase, lipase, and alpha-glucosidase. Plasmids derived from psychrotrophic strains NRRL-B2393 and NRRL-B2397 were introduced into Escherichia coli DH5alpha by electrotransformation, and were shown to be stably maintained. Resistance to ampicillin and cephalothin was conferred, and beta-lactamase and oxidase activity were noted in these transformants. Partial sequencing of pse-2 PCR products from pNRRL-B2393 and pNRRL-B2397, generated nucleotide sequences of 491-bp and 542-bp, and a Basic-Local-Alignment-Search Tool (BLAST ) search produced significantly shared alignments with regions of genomic DNA of E. coli, and with the cytochrome-oxidase ( ccmA, ccmB, and ccmC) genes of Pseudomonas putida P8. These data strongly suggest that plasmid-encoded antibiotic resistance has a common origin, and can be horizontally transferred intra- and intergenerically among species of psychrotrophic Gram-negative bacteria. Moreover, these data demonstrate that plasmid-mediated properties, including antibiotic resistance and enzymatic activity, may be transferred to mesophilic microorganisms. |
