Response of heavy metal and antibiotic resistance genes and related microorganisms to different heavy metals in activated sludge

Highlights

• Metagenomic analysis of Cu and Cr stresses on resistance gene and their related microbe.

• Changes of HMRMs and ARMs are significantly induced by different heavy metals.

• Significant relationship was noted between HMRGs and ARGs and their related microbial community.

• Microbial communities are the main factor driving the diversity and abundance of HMRGs and ARGs.

Abstract

With the recent growing interest of antibiotic resistance genes (ARGs) and their co-selection with heavy metal resistance genes (HMRGs), their relationship to heavy metals needs further analysis. This study examined the response of heavy metal resistant microorganisms (HMRMs) and antibiotic resistant microorganisms (ARMs) and their resistance genes (HMRGs and ARGs) to Cu and Cr stresses using metagenome. Results showed that Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and Nitrospirae are the dominant HMRMs and ARMs, with majority of HMRMs taxa presenting changes similar to ARMs under heavy metal stresses. Types of HMRGs and ARGs changed (increased or decreased) under Cu and Cr stresses, and a significant relationship was noted between HMRGs and ARGs and their related microbe (p < 0.05). Network analysis revealed synergistic relationships between majority of HMRGs and ARGs; however, negative correlations were also noted between them. Co-occurrence of HMRGs and ARGs was mainly observed in chromosomes, and plasmids were found to provide limited opportunities for heavy metals to promote antibiotic resistance through co-selection. These findings imply that the response of HMRMs and ARMs is induced by heavy metals, and that the changes in these microbial communities are the main factor driving the diversity and abundance of HMRGs and ARGs.

Introduction

Antibiotics are widely used in human medicine, animal disease control, and animal husbandry (Kalia et al., 2019; Levy and Marshall, 2004). The widespread prevalence of antibiotics stimulates the growth of antibiotic-resistant microorganisms (ARMs) and antibiotic resistance genes (ARGs) in the environment, which further increases the number of resistant pathogenic microorganisms in the different environment, thus endangering human health (Berendonk et al., 2015). Antibiotics and ARGs from various sources eventually flow into the municipal wastewater treatment plants (WWTPs), where abundant nutrients and high density of microorganisms provide favorable conditions for the horizontal transfer and enrichment of ARGs (Guo et al., 2017; Karkman et al., 2018; Rizzo et al., 2013). Besides, WWTPs are also considered as hot spots of horizontal gene transfer, which facilitates the spread of ARGs among different bacterial species (Aminov, 2011). The toxicity of heavy metals is an increasingly serious problem, not only because of the discharge of heavy metals into the environment (Iftikhar et al., 2021; Tauqeer et al., 2021a, 2021b), but also because of the significant reduction of wastewater biological treatment efficiency (Sun et al., 2016, 2019). In WWTPs, when the microbial community is subjected to the selective pressure of heavy metals, the potential co-selection of resistance genes is very high (Di Cesare et al., 2016).

Studies evidence suggests that heavy metals not only have the ability to impose selective pressure on heavy metal resistance genes (HMRGs) but also on ARGs (Di Cesare et al., 2016; Seiler and Berendonk, 2012). Many studies have shown that heavy metals pollution in the environment has a high correlation with the abundance of ARGs (Ji et al., 2012; Knapp et al., 2011; Pal et al., 2015). Knapp et al. demonstrated that Cu lead to copper resistance, and indirectly induced antibiotic resistance of copper resistant microbe (Knapp et al., 2011). Wang et al. studied the effect of chlortetracycline (CTC) and Cu on ARGs during anaerobic fermentation, and indicated that the combination of CTC and Cu had significant selective pressure on ARGs (Wang et al., 2017). Multiple mechanisms have been reported to participate and co-select of antibiotic and heavy metal resistance. One is co-resistance, in which genes with specific resistance phenotypes are co-existed in the common genetic elements, such as transposon, plasmid, and integron. Di Cesare found that some ARGs, HMRGs, and integrons in a sewage treatment plant were highly correlated, and suggested the occurrence of co-selection between HMRGs and ARGs (Di Cesare et al., 2016). Fang reported the co-existence of oqxAB, blaCTX-M, and the pco and sil operons on the same plasmids, and bacteria containing the plasmid showed co-resistance to heavy metals and antibiotics (Fang et al., 2016). The second is cross-resistance, a common resistance mechanism in which bacteria can simultaneously develop resistance to antibiotics and heavy metals. Conroy et al. found that ges coding system is closely related to RND efflux system, which can expel heavy metals and antibiotics out of cells at the same time (Conroy et al., 2010). The third is co-regulated resistance. When bacteria are exposed to selection pressure, they will connect the different processes and produce resistance through stress coordination response (Baker-Austin et al., 2006). Perron et al. proved that the two-component regulatory gene czcR-czcS is related to heavy metal resistance and carbapenem antibiotic resistance of Pseudomonas aeruginosa (Perron et al., 2004).

Although some studies had been conducted on the impact of heavy metals on HMRGs and ARGs, they had been mainly focused on the correlation between heavy metal and specific resistance genes. The existence of chromosomal resistance genes was underestimated, and the response of resistant microbial communities to heavy metals received little attention. Since resistance genes would be integrated into bacterial chromosomes, it can be speculated that the change of microbial community will play an important role in the resistance gene. The rapid propagation of microorganisms in sewage treatment will inevitably affect the species and abundance of resistance genes. In recent years, the development of metagenomic sequencing has provided an effective method for the comprehensive investigation of resistance genes and their related microbial communities. In this study, we analyzed the changes in HMRGs, ARGs, and their related microbial communities under Cu and Cr stresses using metagenomic analysis. The aims of this study were to: (1) determine the changes in HMRMs and ARMs in sludge under different concentrations of Cu and Cr; 2) assess the variations in abundance and diversity of ARGs and HMRGs in activated sludge in the presence of Cu and Cr; and (3) evaluate the correlation among resistant microorganisms, ARGs, and HMRGs under Cu and Cr stresses. This study will promote the understanding of the effects of heavy metals on resistance genes.