Isolation And Cultivation Ofantibiotic-Resistant Bacteria Frompharmaceutical Wastewater And Analysis Oftheir Application Potential

The problem of bacterial resistance due to the indiscriminate and abusive use of antibiotics has become a worldwide environmental problem,posing a serious threat to the ecological environment and human health.It was found that pharmaceutical wastewater treatment plants,as an important barrier to stop the spread of pollutants to the environment,still have a large number of antibiotic-resistant bacteria（ARBs）and antibiotic-resistant genes（ARGs）in all aspects of their wastewater treatment.In order to further analyse the resistance in pharmaceutical wastewater treatment plants,two strains of ARBs were isolated from the sludge of pharmaceutical wastewater treatment plants using traditional isolation and culture methods,and the resistance of the two strains to different antibiotics and heavy metal ions was investigated.The main findings are as follows:（1）Four strains of ARBs were screened from dewatered sludge of pharmaceutical wastewater treatment plants using norfloxacin antibiotics as the sole carbon source.Two strains of ARBs,named N2 and N4,were further screened by preliminary resistance testing for subsequent experiments.16S r RNA gene sequence homology was identified and phylogenetic.The N2 bacteria were identified as Stenotrophomonas acidaminiphila and the N4 bacteria were identified as Acinetobacter lwoffii by phylogenetic tree analysis.（2）Four antibiotics（chloramphenicol,ampicillin sodium,tetracycline hydrochloride and cefazolin）and three heavy metal ions(Cd²⁺,Cu²⁺and Zn²⁺)were selected to determine the minimum inhibitory concentration（MIC）of the two strains,and then the combined resistance of the strains was analysed.The results showed that the MICs of chloramphenicol and cefazolin were 1024 mg·L^-1for both strains,and the MICs of Cu²⁺and Zn²⁺were 512 mg·L^-1for both strains,reflecting the high resistance of both strains to different classes of drugs.The inhibition of tetracycline hydrochloride and ampicillin sodium in combination with Cu²⁺or Cd²⁺,respectively,was then investigated.The results showed that only the combination of ampicillin sodium and Cd²⁺showed a synergistic effect on N2 bacteria,and the inhibition effect of the two drugs was further enhanced after the combination,while the other drug combinations showed additive or irrelevant effects on N2 bacteria;the combination of different antibiotics and heavy metals showed irrelevant or antagonistic effects on N4bacteria,and the addition of heavy metals increased the resistance of N4 bacteria to the antibiotics;The same antibiotic-heavy metal combination showed completely different inhibitory effects on the two strains of bacteria,showing the individual differences between the two strains.（3）In order to further investigate the resistance mechanism and application potential of the ARBs obtained,the whole genome of N4 was sequenced,with a genome size of 3,732,121 bp,coding for 3,359 genes and containing 30 plasmids.The analysis of antibiotic resistance genes（ARGs）resulted in the annotation of multiple ARGs against different classes of antibiotics in the CARD and ARDB databases,which explains the extremely high resistance shown by the strain to multiple drugs in drug sensitivity tests,the enzyme-modified genes for chloramphenicol andβ-lactam antibiotics also demonstrate the biodegradation potential of N4 bacteria for these two classes of antibiotics.In addition,annotation of its functional gene database revealed that the genome of N4 bacteria contains a large number of genes for inorganic ion transport and metabolism,genes responsible for defence mechanisms,genes for metabolic and enzymatic reactions and biodegradation and metabolism of exogenous substances,the presence of these genes further confirms the potential of N4 bacteria for the remediation of antibiotics or other related contaminants.