Impact of low-dose ozone nanobubble treatments on antimicrobial resistance genes in pond water

Abstract

Antimicrobial resistance (AMR) poses a significant global health threat as the silent pandemic. Because of the use of antimicrobials in aquaculture systems, fish farms may be potential reservoirs for the dissemination of antimicrobial resistance genes (ARGs). Treatments with disinfectants have been promoted to reduce the use of antibiotics; however, the effect of these types of treatments on AMR or ARGs is not well known. This study aimed to evaluate the effects of low dose ozone treatments (0.15 mg/L) on ARG dynamics in pond water using metagenomic shotgun sequencing analysis. The results suggested that ozone disinfection can increase the relative abundance of acquired ARGs and intrinsic efflux mediated ARGs found in the resistance nodulation cell division (RND) family. Notably, a co-occurrence of efflux and non-efflux ARGs within the same bacterial genera was also observed, with most of these genera dominating the bacterial population following ozone treatments. These findings suggest that ozone treatments may selectively favor the survival of bacterial genera harboring efflux ARGs, which may also have non-efflux ARGs. This study underscores the importance of considering the potential impacts of disinfection practices on AMR gene dissemination particularly in aquaculture settings where disinfectants are frequently used at low levels. Future endeavors should prioritize the evaluation of these strategies, as they may be associated with an increased risk of AMR in aquatic environments.

Keywords: antimicrobial resistance genes; disinfection; efflux mediated ARGs; metagenomic shotgun sequencing; ozone treatments.

FIGURE 1

The results of a Hierarchical cluster analysis on acquired resistance genes revealed three distinct clusters corresponding to the two ozone treatments and the control air treatment groups. Treatments are labeled as, A: ozone macrobubble group (O3MB), B: air macrobubble group (AirMB), C: air nanobubble group (AirNB) and D: ozone nanobubble group (O3NB). The number following is the replicate, followed by the sampling time, pre: pretreatment sample, 24 h: sample after 24 h of the treatment. Our analysis showed all pre-treatment samples and 24 h-post control air treatment samples clustered together in the Cluster 1, and the three samples from the 24 h-post ozone nanobubble treated group were categorized into cluster 2. Cluster 3 was comprised by three samples in 24 h-post ozone macrobubble treated group.

FIGURE 2

(A) Relative abundance of acquired ARGs of each sample across four treatments; (B–G) Relative abundance of specific ARGs before (pre) and 24 h after (24 h) air or ozone treatments. Significant difference in the relative abundance of ARGs between pre and post treatment samples were determined using a Paired T-test and is indicated with *. Only one ARG showed a significant decrease. Y-axis represents relative abundance of each gene. X-axis represents sample name, in which A is ozone macrobubble group (O3MB), B is air macrobubble group (AirMB), C is air nanobubble group (AirNB) and D is ozone nanobubble group (O3NB). The number following is the replicate, followed by the sampling time, pre, pretreatment sample; 24 h, sample after 24 h of the treatment.

FIGURE 3

(A–F) Relative abundance of ARGs grouped by antibiotic classes before (pre) and 24 h after (24 h) air or ozone treatments. Significant difference in the relative abundance of genes in different antibiotic classes between pre and post treatment samples were determined using a Paired T-test and is indicated with *. Only one class showed a in significant decrease; (G) Normalized abundance of antibiotics classes of each sample across four treatments, which showed ARGs associated with multidrug resistance increased and dominated in the community after the ozone treatments. Y-axis represents relative abundance of each antibiotic class. X-axis represents sample name, in which A is ozone macrobubble group (O3MB), B is air macrobubble group (AirMB), C is air nanobubble group (AirNB) and D is ozone nanobubble group (O3NB). The number following is the replicate, followed by the sampling time, pre, pretreatment sample; 24 h, sample after 24 h of the treatment.

FIGURE 4

(A–E) Relative abundance of ARGs grouped by resistance mechanisms before (pre) and 24 h after (24 h) air or ozone treatments. Significant difference in the relative abundance of genes in different resistance mechanisms between pre and post treatment samples were determined using a Paired T-test and is indicated with *. Antibiotic target alteration showed a significant decrease after treatments in all groups; (F) Relative abundance of genes grouped by resistance mechanism in each sample across four treatments, which showed ARGs associated with antibiotic efflux mechanism increased and dominated in the community after the ozone treatments. Y-axis represents relative abundance of genes grouped by mechanism. X-axis represents sample name, in which A is ozone macrobubble group (O3MB), B is air macrobubble group (AirMB), C is air nanobubble group (AirNB) and D is ozone nanobubble group (O3NB). The number following is the replicate, followed by the sampling time, pre, pretreatment sample; 24 h, sample after 24 h of the treatment.

FIGURE 5

Relative abundance of specific AMR gene families for intrinsic efflux ARGs before (pre) and 24 h after (24 h) air or ozone treatments. Significant difference in the relative abundance of genes in different families between pre and post treatment samples were determined using a Paired T-test and is indicated with *. Only genes in the RND family showed a significant increase in relative abundance after ozone treatments, while ABC family decreased significantly after ozone treatments. Y-axis represents relative abundance of genes grouped by mechanism. X-axis represents sample name, in which AirMB is air macrobubble group, AirNB is air nanobubble group, O3MB is ozone macrobubble group and O3NB is ozone nanobubble group. The number following is the replicate, followed by the sampling time, pre, pretreatment sample; 24 h, sample after 24 h of the treatment.

FIGURE 6

Relative abundance of intrinsic efflux ARGs within specific AMR gene families including ATP binding cassette (ABC), resistance nodulation cell division (RND), small multidrug resistance (SMR), and major facilitator super (MFS) family detected by CARD-RGI identifier before (pre) and 24 h after (24 h) air or ozone treatments delivered by macrobubbles or nanobubbles, which showed adeF gene became the most abundant intrinsic efflux ARGs in the water samples 24 h after the ozone treatments. Y-axis represents relative abundance of genes. X-axis represents sample name, in which A is ozone macrobubble group (O3MB), B is air macrobubble group (AirMB), C is air nanobubble group (AirNB) and D is ozone nanobubble group (O3NB). The number following is the replicate, followed by the sampling time, pre, pretreatment sample; 24 h, sample after 24 h of the treatment.

FIGURE 7

Sankey diagram used to investigate whether acquired ARGs or intrinsic efflux ARGs co-existed within the same known genera of bacteria 24 h after air treatment ed samples (A) and ozone treatment (B), while highlighting the interplay of resistance mechanisms. Regardless of the treatment efflux pump ARGs were linked to almost all bacterial genera with the exception of 2 or 3, suggesting ozone treatment did not alter the co-existence pattern of ARGs.

FIGURE 8

The relative abundance of classified bacterial genera with acquired efflux ARGs (left) and intrinsic efflux ARGs (right) in all samples. Y-axis represents relative abundance of bacteria with ARGs. X-axis represents sample name, in which A is ozone macrobubble group; B is air macrobubble group; C is air nanobubble group and D is ozone nanobubble group. The number following is the replicate, followed by the sampling time, pre, pretreatment sample; 24 h, sample after 24 h of the treatment.