Prevalence of diverse antimicrobial resistance genes and bacteria in sewage treatment plant-derived sludge environment

Abstract

Antimicrobial resistance (AMR) contamination in the environment is one of the most significant worldwide threats of the 21^st century. Since sludge is heavily exposed to diverse contaminants, including pharmaceuticals, the inhabitant bacterial population is expected to exhibit resistance to antimicrobial agents. In this study, sewage treatment plant (STP) sludge samples were analyzed to assess the antibiotic-resistant bacterial population, abundance of AMR genes (ermF, qnrS, Sul1, blaGES, blaCTX-M, and blaNDM), and mobile genetic elements (intl1 and IS26). Out of 16, six bacterial isolates exhibited resistance to 13 antibiotics with a high multiple antibiotic resistance index (MARI) (0.93) and high metal tolerance. Quantitative polymerase chain reaction showed the abundance of target genes ranging from 6.6 × 10³ to 6.5 × 10⁸ copies g^-1 sludge. The overall outcome reveals that STP sludge comprised varied multidrug-resistant bacterial populations. It will give insights into the functions of heavy metals and biofilm development in the selection and spread of AMR genes and the associated bacteria. Therefore, the application of sludge needs proper screening for AMR and metal contamination prior to its countless applications. This study will contribute immensely to the risk analysis of STP effluents on environmental health, including control of AMR transmission.

Keywords: antimicrobial resistance gene; biofilm; metal resistance; multi-drug resistance; sewage treatment plant; sludge.

Figure 1.

Percentage of resistance for selected ampicillin-resistant bacterial isolates for STP sludge samples with 14 different tested antibiotics. (A) percentage of resistant isolates for STP1 sludge, (B) a percentage of resistant isolates for STP2, (C) a percentage of resistant isolates for STP 3, and (D) based on the resistance multiple antibiotic resistance index (MARI) is represented for three STPs.

Figure 2.

Neighbor-joining tree based on distance analysis representing the relationship between the 16S rRNA sequences of 16 bacterial isolates from three different STPs sludge samples and 48 reference sequences (16S rRNA gene) of the related species from NCBI GenBank. Bootstrap values generated from 500 replicates are shown at the nodes.

Figure 3.

Quantitative real-time polymerase chain reaction (qRT-PCR) for the enumeration of six antibiotic resistance genes (ARGs) (ermF, qnrS, Sul1, blaNDM, blaGES, and blaCTX) and two mobile genetic elements (MGEs) (intl1 and IS26) along with 16S rRNA gene in the three STPs sludge samples. Values (log-transformed) are expressed for the gene copy number per gram of the dry weight of sludge, normalized to the DNA extraction yield.

Figure 4.

The box plot represents the first and third quartile of the six antibiotic resistance genes (ARGs) (A) ermF, (B) qnrS, (C) Sul1, (D) blaGES, (E) blaCTX, (F) blaNDM, and two mobile genetic elements (MGEs) (G) and intl1 (H) IS26 target genes for three STP sludge samples.

Figure 5.

Principal component analysis (PCA) showing the correlations of ARGs and MGEs gene abundance and different STPs with (A) the physico-chemical parameters denoted as BD = bulk density, WHC = water holding capacity, M = moisture, pH, OC = organic carbon, TN = total nitrogen, AN = available nitrogen, AP = available phosphorus, and EC = electrical conductivity, with antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) gene abundance, and different STPs (STP1–Bhagwanpur 8 MLD, STP2–Dinapur 80 MLD, and STP3–Dinapur 140 MLD), and (B) Metals denoted as 1 = Fe, 2 = Cu, 3 = Zn, 4 = Mn, 5 = Cd, 6 = Cr, 7 = Ni, and 8 = Pb.