| Shandong is one of the most prominent provinces for livestock and poultry breeding in China.In recent years,the livestock and poultry breeding industry has rapidly developed.This,however,is inevitably accompanied by a large number of emissions of breeding pollutant.In particular,a large amount of wastewater that contains pollutants such as Escherichia coli（E.coli）pathogen and heavy metals（Heavy metal,HM）is produced during the breeding of large animals such as swine and cattle,posing a great threat to public health.In this study,47 conventional dairy farms and 48 conventional pig farms in Shandong province were selected as sampling sites to collect breeding wastewater.The E.coli was isolated from wastewater samples and identifying this by the use of a microbial mass spectrometer.Subsequently,the sensitivity test of E.coli to 29 different antibiotics in ten categories was measured using the minimum inhibitory concentration（MIC）method.PCR was used to detect the common antibiotic resistance genes（ARGs）in E.coli and the resistance genes of quaternary ammonium compound（QAC）disinfectants.Finally,the multiplex PCR was used to determine Clermont phylogenetic types of E.coli.The contents of copper（Cu）,zinc（Zn）,cadmium（Cd）,lead（Pb）,arsenic（As）,selenium（Se）,nickel（Ni）and iron（Fe）in the samples were determined by inductively coupled plasma mass spectrometry（ICP-MS）.The contents of mercury（Hg）in the samples were determined by atomic fluorescence spectrometry（AFS）and the content of hexavalent chromium（Cr（VI））in wastewater samples was determined using the diphenylcarbazide method.Combined with the relevant existing limits of HM content in surface water in China,the comprehensive pollution index（P）of each HM element was calculated.The results are provided in the following sections.A total of 54 E.coli strains were isolated from dairy farms’ wastewater samples.Drug sensitivity results showed that 17 strains（31.5%,17/54）were resistant to 14 common antibiotics in five categories,with the highest resistance rate of 25.9%（14/54）to tetracycline（TE）.In terms of drug types,E.coli were mainly resistant to tetracyclines（82.4%,14/17）andβ-lactams（70.6%,12/17）antibiotics.For E.coli,22.22%（12/54）were multidrug-resistant（MDR）strains,with 9 drug resistance profiles,the main resistance profile was ATM + CZ +FEP + CRO + CXM,distributed in Tai’an and Zibo breeding wastewater.A total of eight kinds of ARGs in five categories were detected,the highest of which was 20.4%（11/54）for tet A,followed by qnr S（16.7%,9/54）.The detection rate of ARGs in tetracyclines was the highest（24.1%,13/54）.A total of 13.0%（7/54）of E.coli carried three or more kinds of ARGs.The genome-encoded QAC disinfectant resistance genes could be detected in all E.coli strains,and the the detection rate of mobile plasmid-encoded QAC disinfectant resistance genes was 1.9%.Phylogenetic analysis showed that 48.1% of E.coli were genotype A,44.2%were genotype B1,1.9% were genotype C,and 1.9% were genotype F,the genotypes of 5.6%of E.coli were unknown.The phylogenetic types of MDR E.coli were mainly A（33.3%,4/12）and B1（50%,6/12）.A total of 45 E.coli strains were isolated from swine farms’ wastewater samples.Drug susceptibility results showed that 43 strains（95.6%,43/45）were resistant to 20 common antibiotics in nine categories,with the highest resistance rate of 91.1%（41/45）to TE.The E.coli were mainly resistant to tetracyclines（95.3%,41/43）and chloramphenicol（81.4%,35/43）antibiotics.A total of 31 E.coli were MDR strains and distributed in swine farm wastewater of every cities in Shandong province,with 24 drug resistance profiles,the dominant resistance profile was C + TE + SMX,distributed in Heze,Jining,Yantai,Linyi and Tai’an breeding wastewater.A total of sixteen kinds of ARGs in six categories were detected,the highest of which was 77.8%（35/45）for qnr S,followed by tet A（66.7%,30/45）,aminoglycosides resistant gene aad A（62.2%,28/45）and quinolones resistant gene qnr A（62.2%,28/45）.The detection rate of ARGs in quinolones was the highest（95.6%,43/45）,followed by tetracyclines（88.9%,40/45）.A total of 91.1%（41/45）of E.coli carried three or more kinds of ARGs.The detection rate of QAC disinfectant resistant genes was 100%,and the detection rates of mobile plasmid encoded qac E,qac EΔ1,qac F,qac G and sug E（p）were 0%,15.6%（7/45）,37.8%（17/45）,0% and 0%,respectively.Phylogenetic analysis showed that genotype A accounted for 84.4%,B1 for 11.1%,and genotypes of two strains were unknown.The phylogenetic types of MDR E.coli were mainly A（93.5%,29/31）.The maximum pollution index（Imax）of Fe in dairy farm wastewater was 1.97,indicating pollution risk,the P value of other elements were less than 0.6,which were in the ideal level.And Zn（P = 3.13）and Fe（P = 708.9）in swine breeding wastewater were severely polluted.The most serious Fe pollution was mainly distributed in southern Shandong,northern Shandong and part of east Shandong.The contents of As in the two sampling sites of Dezhou and Weifang exceeded the standard,and the contents of Se in two sampling sites,Dongying and Weihai,exceeded the standard,both of which had potential pollution risks.In addition,we also found that Fe pollution had no significant effect on the antibiotic resistance rates and ARGs detection rates of E.coli in wastewater.In this study,the characteristics of antibiotic resistant E.coli and HM residues pollutants in swine and dairy wastewater in Shandong province were studied,and the potential health risks of HM and antibiotic resistant E.coli were evaluated.It is expected to provide a guidance for the purification of a breeding environment,rational usage of mineral element and antibiotic additives,prevention and control of colibacillosis,and promotion of the sustainable development of a green,healthy,and safe livestock and poultry breeding industry,which has important significance to environmental management and public health. |
PDF Full Text Request