Distribution and Classification of Serine β-Lactamases in Brazilian Hospital Sewage and Other Environmental Metagenomes Deposited in Public Databases

Abstract

β-lactam is the most used antibiotic class in the clinical area and it acts on blocking the bacteria cell wall synthesis, causing cell death. However, some bacteria have evolved resistance to these antibiotics mainly due the production of enzymes known as β-lactamases. Hospital sewage is an important source of dispersion of multidrug-resistant bacteria in rivers and oceans. In this work, we used next-generation DNA sequencing to explore the diversity and dissemination of serine β-lactamases in two hospital sewage from Rio de Janeiro, Brazil (South Zone, SZ and North Zone, NZ), presenting different profiles, and to compare them with public environmental data available. Also, we propose a Hidden-Markov-Model approach to screen potential serine β-lactamases genes (in public environments samples and generated hospital sewage data), exploring its evolutionary relationships. Due to the high variability in β-lactamases, we used a position-specific scoring matrix search method (RPS-BLAST) against conserved domain database profiles (CDD, Pfam, and COG) followed by visual inspection to detect conserved motifs, to increase the reliability of the results and remove possible false positives. We were able to identify novel β-lactamases from Brazilian hospital sewage and to estimate relative abundance of its types. The highest relative abundance found in SZ was the Class A (50%), while Class D is predominant in NZ (55%). CfxA (65%) and ACC (47%) types were the most abundant genes detected in SZ, while in NZ the most frequent were OXA-10 (32%), CfxA (28%), ACC (21%), CEPA (20%), and FOX (19%). Phylogenetic analysis revealed β-lactamases from Brazilian hospital sewage grouped in the same clade and close to sequences belonging to Firmicutes and Bacteroidetes groups, but distant from potential β-lactamases screened from public environmental data, that grouped closer to β-lactamases of Proteobacteria. Our results demonstrated that HMM-based approach identified homologs of serine β-lactamases, indicating the specificity and high sensitivity of this approach in large datasets, contributing for the identification and classification of a large number of homologous genes, comprising possible new ones. Phylogenetic analysis revealed the potential reservoir of β-lactam resistance genes in the environment, contributing to understanding the evolution and dissemination of these genes.

Keywords: Hidden-Markov-Model; hospital sewage; metagenome; phylogenetic diversity; serine β-lactamases.

FIGURE 1

Taxonomical distribution of the most predominant groups of Bacteria Phylum present in South Zone (SZ) and North Zone (NZ) samples from Rio de Janeiro (Brazil).

FIGURE 2

Abundance (%) of serine β-lactamase classes (A,C,D) screened by the pHMM-based approach of SZ and NZ hospital sewage samples of Rio de Janeiro (Brazil). Abundance means the percentage of reads related to a specific β-lactamase class/total of reads of each metagenomes sample.

FIGURE 3

Relative abundance (%) of Class A serine β-lactamases screened from SZ and NZ hospital sewage samples of Rio de Janeiro (Brazil). Relative abundance means the percentage of reads related to each β-lactamase type or variant/total of Class A reads for each sample.

FIGURE 4

Relative abundance (%) of Class C serine β-lactamases screened from SZ and NZ hospital sewage samples of Rio de Janeiro (Brazil). Relative abundance (%), means the percentage of reads related to each β-lactamase type or variant/total of Class C reads for each sample.

FIGURE 5

Relative abundance (%) of Class D serine β-lactamases screened from SZ and NZ hospital sewage samples of Rio de Janeiro (Brazil). Relative abundance means the percentage of reads related to each β-lactamase type or variant/total of Class D reads for each sample.

FIGURE 6

Phylogeny of Class A β-lactamase retrieved from hospital sewage metagenomes of SZ and NZ of Rio de Janeiro (Brazil). Representative β-lactamase sequences are indicated by a black closed circle indicates and hospital sewage sequences are indicated by a green closed triangle. Black closed triangle represents the outgroup (Class D) composed of many curated sequences collapsed. The phylogenetic tree was calculated by RAxML-HPC program, using Maximum Likelihood and WAG as the substitution model. The bootstrap value was 100 iterations were used and its values can be observed on the branches (above 50%). Scale bar indicates 0.5 amino acid substitutions per site. ZS stands for SZ (South Zone) and ZN stands for NZ (North Zone). (B) Phylogeny of Class A β-lactamase retrieved from public databases metagenomes (CAMERA and IMG/M). Representative β-lactamase sequences are indicated by a black closed circle and hospital sewage sequences are indicated by a green closed triangle. Black closed triangle represents the outgroup (Class D) composed of many curated sequences collapsed. The phylogenetic tree was calculated by RAxML-HPC program, using Maximum Likelihood and WAG as the substitution model. Bootstrap values (%), based on 100 iterations, can be observed on the branches (above 50%). Scale bar indicates 0.5 amino acid substitutions per site.

FIGURE 7

Phylogeny of Class C β-lactamase retrieved from hospital sewage metagenomes of SZ and NZ of Rio de Janeiro (Brazil), and from public databases (CAMERA/IMG/M). Representative β-lactamase sequences obtained from RefSeq database are indicated by a blue closed square, sequences from public databases are indicated by a closed green triangle, and hospital sewage sequences of Rio de Janeiro are indicated by a black closed circle. Black closed triangle represents the outgroup (Class D) composed of many curated sequences collapsed. The phylogenetic tree was calculated by RAxML-HPC program, using Maximum Likelihood and WAG as the substitution model. Bootstrap values (%), based on 100 iterations, can be observed on the branches (above 50%). Scale bar indicates 0.5 amino acid substitutions per site. ZS stands for SZ (South Zone) and ZN stands for NZ (North Zone).

FIGURE 8

Phylogeny of Class D β-lactamase retrieved from hospital sewage metagenomes of SZ and NZ of Rio de Janeiro (Brazil), and from public databases (CAMERA/IMG/M). Representative β-lactamases sequences obtained from RefSeq database are indicated by a blue closed square, public databases sequences are indicated by a closed green triangle, and hospital sewage sequences of Rio de Janeiro are indicated by a black closed circle. Black closed triangle represents the outgroup (Class C) composed of many curated sequences collapsed. The phylogenetic tree was calculated by RAxML-HPC program, using Maximum Likelihood and WAG as the substitution model. Bootstrap values (%), based on 100 iterations, can be observed on the branches (above 50%). Scale bar indicates 0.5 amino acid substitutions per site. ZS stands for SZ (South Zone) and ZN stands for NZ (North Zone).