High diversity of extended-spectrum beta-lactamase-producing bacteria in an urban river sediment habitat

Abstract

Antibiotic-resistant bacteria (ARB) have been surveyed widely in water bodies, but few studies have determined the diversity of ARB in sediment, which is the most taxon-abundant habitat in aquatic environments. We isolated 56 extended-spectrum beta-lactamase (ESBL)-producing bacteria from a single sediment sample taken from an urban river in China. All strains were confirmed for ESBL-producing capability by both the clavulanic acid combination disc method and MIC determination. Of the isolated strains, 39 were classified as Enterobacteriaceae (consisting of the genera Escherichia, Klebsiella, Serratia, and Aeromonas) by 16S rRNA gene sequencing and biochemical analysis. The present study identifies, for the first time, ESBL-producing strains from the families Brucellaceae and Moraxellaceae. The bla(CTX-M) gene was the most dominant of the ESBL genes (45 strains), while the bla(TEM) gene was the second-most dominant (22 strains). A total of five types of bla(CTX-M) fragments were identified, with both known and novel sequences. A library of bla(CTX-M) cloned from the sediment DNA showed an even higher diversity of bla(CTX-M) sequences. The discovery of highly diverse ESBL-producing bacteria and ESBL genes, particularly bla(CTX), in urban river sediment raises alarms for potential dissemination of ARB in communities through river environments.

FIG. 1.

Phylogenetic tree of 16S rRNA of ESBL-producing isolates. The tree was rerooted with Bacillus subtilis (EU755383) as the root. The branch numbers refer to the percent confidence as estimated by a bootstrap analysis with 100 replications. Scale bar indicates percent divergence. (The first letter of strain names, C or A, indicates the antibiotics used for enrichment, with C for cefotaxime and A for ampicillin. The second word of strain names indicates the isolation medium.)

FIG. 2.

Antibiotic resistance percentages for 56 isolated strains. y axis, percentage of resistant and intermediate (R+I) strains among all tested bacteria.

FIG. 3.

Dendrogram tree of bla_CTX-M gene sequences. bla_CTX-M types A to E were identified from the cultured bacteria, and colonies were isolated from the library. The number in parentheses shows the number of times the sequence was determined in the isolated bacterium or library. The branch numbers refer to the percent confidence as estimated by a bootstrap analysis with 100 replications.