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Isolation and characterization of integron-containing bacteria without antibiotic selection.

ABSTRACT: The emergence of antibiotic resistance among pathogenic and commensal bacteria has become a serious problem worldwide. The use and overuse of antibiotics in a number of settings are contributing to the development of antibiotic-resistant microorganisms. The class 1 and 2 integrase genes (intI1 and intI2, respectively) were identified in mixed bacterial cultures enriched from bovine feces by growth in buffered peptone water (BPW) followed by integrase-specific PCR. Integrase-positive bacterial colonies from the enrichment cultures were then isolated by using hydrophobic grid membrane filters and integrase-specific gene probes. Bacterial clones isolated by this technique were then confirmed to carry integrons by further testing by PCR and DNA sequencing. Integron-associated antibiotic resistance genes were detected in bacteria such as Escherichia coli, Aeromonas spp., Proteus spp., Morganella morganii, Shewanella spp., and urea-positive Providencia stuartii isolates from bovine fecal samples without the use of selective enrichment media containing antibiotics. Streptomycin and trimethoprim resistance were commonly associated with integrons. The advantages conferred by this methodology are that a wide variety of integron-containing bacteria may be simultaneously cultured in BPW enrichments and culture biases due to antibiotic selection can be avoided. Rapid and efficient identification, isolation, and characterization of antibiotic resistance-associated integrons are possible by this protocol. These methods will facilitate greater understanding of the factors that contribute to the presence and transfer of integron-associated antibiotic resistance genes in bacterial isolates from red meat production animals.

SUBMITTER: Barlow RS

PROVIDER: S-EPMC353070 | biostudies-literature | 2004 Mar

REPOSITORIES: biostudies-literature

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Isolation and characterization of integron-containing bacteria without antibiotic selection.

Barlow Robert S RS Pemberton John M JM Desmarchelier Patricia M PM Gobius Kari S KS

Antimicrobial agents and chemotherapy 20040301 3

The emergence of antibiotic resistance among pathogenic and commensal bacteria has become a serious problem worldwide. The use and overuse of antibiotics in a number of settings are contributing to the development of antibiotic-resistant microorganisms. The class 1 and 2 integrase genes (intI1 and intI2, respectively) were identified in mixed bacterial cultures enriched from bovine feces by growth in buffered peptone water (BPW) followed by integrase-specific PCR. Integrase-positive bacterial co ...[more]

PMID: 14982773

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Project description:BACKGROUND:Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, are now accepted as suitable alternatives to antibiotics in the control of animal infections and improving animal production. Lactic acid bacteria (LAB) with remarkable functional properties have been evaluated in different studies as possible probiotic candidates. The purpose of this study was to isolate, characterize and assess the potentials of LAB from poultry gastrointestinal tract as potential poultry probiotics. RESULTS:Potential LAB probiotics were isolated from broilers, characterized and evaluated for probiotic properties including antagonistic activity (against Escherichia coli, E. coli O157: H7, Enterococcus faecalis, Salmonella Typhimurium, S. Enteritidis and Listeria monocytogenes), survivability in simulated gastric juice, tolerance to phenol and bile salts, adhesion to ileum epithelial cells, auto and co-aggregation, hydrophobicity, ?-glucosidase inhibitory activity, and antibiotic susceptibility tests. Most promising LAB strains with excellent probiotic potentials were identified by API 50 CHL and 16S rRNA sequencing as Lactobacillus reuteri I2, Pediococcus acidilactici I5, P. acidilactici I8, P. acidilactici c3, P. pentosaceus I13, and Enterococcus faecium c14. They inhibited all the pathogens tested with zones of inhibition ranging from 12.5?±?0.71 to 20?±?0?mm, and competitively excluded (P?<?0.05) the pathogens examined while adhering to ileum epithelial cells with viable counts of 3.0 to 6.0 Log CFU/ml. The selected LAB strains also showed significant (P?<?0.005) auto and co-aggregation abilities with ?-glucosidase inhibitory activity ranging from 12.5 to 92.0%. The antibiotic susceptibility test showed 100.00% resistance of the LAB strains to oxacillin, with multiple antibiotic resistance indices above 0.5. CONCLUSION:The selected LAB strains are ideal probiotic candidates which can be applied in the field for the improvement of poultry performance and control of pathogens in poultry, hence curtailing further transmission to humans.

Project description:Four streptomycin-resistant isolates of Xanthomonas oryzae pv. oryzae (YNA7-1, YNA10-2, YNA11-2, and YNA12-2) were examined via PCR amplification for the presence of class 1, class 2, and class 3 integrons and aadA1 and aadA2 genes, which confer resistance to streptomycin and spectinomycin. The class 1 integrase gene intI1 and the aminoglycoside adenylyltransferase gene aadA1 were identified in all four resistant isolates but not in 25 sensitive isolates. PCR amplifications showed that 7790-bp, 7162-bp, 7790-bp, and 7240-bp resistance integrons with transposition gene modules (tni module) in 3' conserved segments existed in YNA7-1, YNA10-2, YNA11-2, and YNA12-2, respectively. Subsequent analysis of sequences indicated that the integrons of YNA7-1 and YNA11-2 carried three gene cassettes in the order |aacA3|arr3|aadA1|. The integron of YNA10-2 carried only |arr3|aadA1| gene cassettes. The integron of YNA12-2 lacked a 550-bp sequence including part of intI1 but it still carried |aacA3|arr3|aadA1| gene cassettes. The analysis of inactive mutants and complementation tests confirmed that the aacA3 gene conferred resistance to tobramycin, kanamycin, gentamicin and netilmicin; the arr3 gene conferred resistance to rifampicin; and the aadA1 gene conferred resistance to streptomycin and spectinomycin. The resistance phenotypes of the four isolates corresponded with their resistance gene cassettes, except that YNA7-1 and YNA12-2 did not show rifampicin resistance. Sequence comparison revealed that no gene cassette array in GenBank was in the same order as in the integrons of the four resistant isolates in this study and the aadA1, which was identical in the four resistant isolates, showed 99% identity with aadA1 sequences in GenBank. The result of a stability test showed that the resistance phenotype, the aadA1 gene, and the intI1 gene were completely stable in YNA7-1 and YNA12-2 but unstable in YNA10-2 and YNA11-2. To our knowledge, this is the first report of resistance integron in a phytopathogenic bacteria.

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Isolation and characterization of integron-containing bacteria without antibiotic selection.

Publications

Isolation and characterization of integron-containing bacteria without antibiotic selection.

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