Antibiotic Resistant Bacteria and Genes in Bioaerosols at Wastewater Treatment Plant: Should I Be Worried?

Background Air is potentially an important exposure pathway for antibiotic resistance genes (ARGs) and bacteria (ARBs) in some environments (1). However, most studies on environmental antibiotic resistance focus on water, leaving air as an understudied matrix (2, 3). There is more to learn about the sources, nature and human health risks of the airborne antibiotic resistance (as reviewed in 4). Specifically, most studies focus on air as an isolated matrix, overlooking the interrelationships with antibiotic resistance in the other surrounding environmental matrices such as water, soil, animals, or insects (5). WWTPs are a source of ARG emissions to the atmosphere (3), and understanding the concentrations of and therefore potential exposure to ARBs via bioaerosols at wastewater treatment plants (WWTPs) is of particular interest given the immediate hazard posed. Moreover, the ARB methicillin resistant Staphylococcus aureus (MRSA) is not only a major cause of hospital-acquired infections, but also community-acquired infections (6). This antibiotic resistant opportunistic pathogen can be transmitted to humans through humans, fomites, and, most importantly for the present study, air (7). Objectives The objectives of this study were to a) detect the presence of methicillin resistant Staphylococcus aureus in air from the grit chamber and UV room of a WWTP, b) compare the concentrations of select ARGs in water and bioaerosols at these locations and b) study the resistome, microbiome, and mobilome of air samples in comparison to paired wastewater influent and effluent samples. A combination of culturing, qPCR, and metagenomic sequencing were used in this study. Methodology Samples were collected from two locations (grit and UV chamber) at a tertiary WWTP. The treatment train includes bar screens, grit chamber, primary clarification, trickling filtration, secondary clarification, aeration basins for nitrification, final clarifiers, pressure filtration, and UV disinfection. Air samples were collected in replicates using the stationary Electrostatic Bioaerosol Sampler (SEBS, 8) at 20 L/min. Samplers were operated for 7.5 hours and samples were collected on five days at each site in fall 2022. Paired composite water samples (influent and effluent) were collected at the time of air sampling. Air and water samples were split for biomolecular (qPCR and metagenomics on total samples) and cultivation-based analyses (heterotrophs via TSA and MRSA via CHROMAgar MRSA). All of the colonies growing on TSA were also analyzed via qPCR for select ARGs. Results Similar concentrations of cultivable bacteria (reported as CFUs/m3) were observed for air samples collected from the grit chamber and UV-effluent site (paired Wilcoxon test, p= 0.4). While CFUs were measurable on most days (N=4/5) in air samples from UV disinfection room, the paired disinfected water samples (N=2/5) did not necessarily show growth on TSA. To understand the potential hazard posed by the cultivable bacteria, ARGs were quantified in the colonies collected from the TSA agar plates. For air samples, the colonies collected from TSA plates contained qacE genes, encoding for quaternary ammonium compound resistance, at both sites in most of the samples (N=4/5, 80%). The genes sul1, blaTEM, and tet(G) were detected in only one sample, encoding for sulfonamide, beta lactam, and tetracycline resistance. In water samples, all of the four targeted ARGs were detected and quantified in four of the influent samples. These ARGs were generally not detected in effluent water samples. One antibiotic resistant pathogen was cultivated in the present study: MRSA. MRSA was detected on four days at the grit-chamber and two days in air samples at the UV-effluent. MRSA was not previously reported in air sampled using different techniques at a WWTP from a study conducted in Poland (9). MRSA was detected on four days in the wastewater influent samples and in none of the effluent samples. For samples analyzed via direct biomolecular analysis, bacterial 16S rRNA was detected in all samples. As expected, sul1 was detected in all influent and effluent samples, it was detected only in three air samples (Fig. 1). The three ARGs detected via metagenomics analysis in the air were tet(K), mdtB and muxB encoding for tetracycline, aminocoumarin, and multidrug resistance (Table 1). mdtB and tet(K) were detected in both air and water paired samples. The ARGs detected in air represent a very small portion of the ARGs observed in the paired wastewater where hundreds of ARGs were annotated in each sample. Significance Protecting public health of utility operators requires considering the potential hazards posed by various pathogen exposure routes. These data presented will help answer some questions about the relationship between bioaerosols and wastewater at two stages of treatment.