High Diversity of CTX-M Extended-Spectrum β-Lactamases in Municipal Wastewater and Urban Wetlands

Abstract

The CTX-M-type extended-spectrum β-lactamases (ESBLs) present a serious public health threat as they have become nearly ubiquitous among clinical gram-negative pathogens, particularly the enterobacteria. To aid in the understanding and eventual control of the spread of such resistance genes, we sought to determine the diversity of CTX-M ESBLs not among clinical isolates, but in the environment, where weaker and more diverse selective pressures may allow greater enzyme diversification. This was done by examining the CTX-M diversity in municipal wastewater and urban coastal wetlands in southern California, United States, by Sanger sequencing of polymerase chain reaction amplicons. Of the five known CTX-M phylogroups (1, 2, 8, 9, and 25), only genes from groups 1 and 2 were detected in both wastewater treatment plants (WWTPs), and group 1 genes were also detected in one of the two wetlands after a winter rain. The highest relative abundance of blaCTX-M group 1 genes was in the sludge of one WWTP (2.1 × 10(-4) blaCTX-M copies/16S rRNA gene copy). Gene libraries revealed surprisingly high nucleotide sequence diversity, with 157 new variants not found in GenBank, representing 99 novel amino acid sequences. Our results indicate that the resistomes of WWTPs and urban wetlands contain diverse blaCTX-M ESBLs, which may constitute a mobile reservoir of clinically relevant resistance genes.

FIG. 1.

Distribution of bla_CTX-M group 1 cloned gene sequences in each library grouped according to the most similar reference sequence variants. Only validated bla_CTX-M sequences from www.lahey.org/Studies/ were used as references. TRE, Tijuana River Estuary; PLE, Point Loma Wastewater Treatment Plant effluent; SBI, South Bay International Wastewater Treatment Plant. Color images available online at www.liebertpub.com/mdr

FIG. 2.

Shannon entropy of each amino acid position calculated across each library and among all CTX-M group 1 reference sequences from the Lahey Clinic (www.lahey.org/Studies/). Bars greater than zero indicate the presence of two or more alleles, with greater height of the bar generally indicating greater frequency of the rare allele and rarely the presence of more than two alleles in the library. Solid icons indicate amino acid positions with variability shared with the CTX-M group 1 reference sequences. Figure created using R (R Foundation for Statistical Computing, Vienna, Austria [www.R-project.org/]). Amino acid numbering is absolute (i.e., not referenced to S70 and position 239 is included). Color images available online at www.liebertpub.com/mdr

FIG. 3.

Venn diagram of variable amino acid positions shared between environmental bla_CTX-M gene libraries. Figure created using Venny (Venny. An interactive tool for comparing lists with Venn diagrams [bioinfogp.cnb.csic.es/tools/venny/index.html]). Color images available online at www.liebertpub.com/mdr