Effect Of Co-selection And Genome Plasticity On Bacterial Adaptation To High Level Of Contaminants

The high frequency of antibiotic resistance being an environmental pollution is a global public health concern.Environmental selection of antibiotic resistance genes(ARGs)is considered to be due to antibiotic residues or metal residues,extensively used in livestock.In this study,we selected three commercial poultry farms to compare the co-occurrence patterns of antibiotics and metals residues in relation to ARGs.We also validated a method to determine the antibiotics concentration using LCMS-MS.1.A simultaneous extraction and cleanup method was optimized and validated for the determination of multiclass antibiotics in sediments by liquid chromatography with tandem quadruple mass spectrometry(LC-MS/MS).The method was optimized for 40 antibiotics ranging from cephalosporin,fluoroquinolone,lincosamide,macrolide,nitroimidazole,quinolone,sulfonamide,and tetracycline groups.The method involved hydration of freeze dried sediment samples with 5 ml McIlvaine buffer followed by extraction by using a 10 ml mix of MeOH and MeCN(1:3 v/v).The liquid-liquid partitioning between aqueous and organic extract phases was achieved by salting out with MgSO4(4 g)and NaCl(1 g)and centrifugation at 10,000 rpm for 5 minutes.An aliquot(1ml)of organic extract was subjected to dispersive solid phase extraction by using 100 mg mix of C18 and PSA(1:2 w/w)and 50 mg MgSO4 followed by filtration through 0.22?m syringe filters prior to LC-MS/MS analysis.The method was validated for 10,20,50 and 100 ?g kg-1 spiking levels by using blank sediment samples obtained from a drinking water reservoir according to the guidelines of European Commission Decision(2002)2002/657/EC.The method produced clean extracts with generally low matrix effect during LC-MS/MS analysis.The recoveries of the antibiotics ranged between 24-162%,31-151%,35-176%and 35-188%for 10,20,50 and 100 ?g kg-1 spiking levels with acceptable precision(RSD>20%)in the majority of cases.The analytical method was sensitive to achieve 0.004-7.7 ?gml-1 and 0.01-25.6 ?gml-1 limits of detection and quantitation,respectively.The scope of the method was investigated by analyzing complex solid matrices(chicken manure,poultry feed and soil)spiked at 10,20,50 and 100 ?g kg-1 levels with desirable accuracy and precision.The method was also applied for the antibiotic analysis in samples with incurred residues.Residues of fluoroquinolone,macrolide,sulfonamide and tetracycline antibiotics were detected in the sediment,chicken manure,soil and poultry feed samples.2.Poultry feed with metal additives is polluting the environment by providing a long-term co-selective pressure for antibiotic resistance.To help strengthen this hypothesis we determined the heavy metal concentrations of Zn,Cu,As,Ni,Cd,Pb,and Cr,from poultry farm samples using ICP-MS and data was analyzed using Pearson's correlation matrix.Concentration of Zn in soil was found to be highest in all farms,ranging from 39.05-123.37 mg kg-1,to 46.17-394.47 mg kg-1,and 61.39-990.57 mg kg-1 respectively,followed by Pb in farm "B" at 33.44-618.22 mg kg-1 and in farm "C" at 111.08 mg kg-1.The contents of Zn,Cu,Cd and Ni in feed samples with maximum ranges were of 103.09-304.29 mg kg-1,6.48-178.87 mg kg-1,4.15-5.46 mg kg-1 and 3.35-4.53 mg kg-1 which is alarming.In manure,the contents of Zn were found to be much higher at 287.29 mg kg-1-1103.09 mg kg-1 followed by Cu at 28.64 mg kg-1-273.25 mg kg-1 and Cd at 0.46 mg kg-1-16.36 mg kg-1 respectively in farm "B" The contents of Cr,Ni and Pb were all in safe ranges.A strong correlation between Ni and Cr(r=0.983,P<0.01)was found among the 39 soil samples in Pearson's correlation matrix.The correlation between Zn and As(r?0.749,P<0.01)and Zn and Cu(r=0.684,P<0.01)among 42 feed samples were significantly positive.In manure,a significant positive correlation between Zn and Cu(r=.846,P<0.01)and between Ni and Cu(r=0.874,P<0.01)was detected.These results indicate the high levels of metals in poultry feed causing environmental pollution.3.Antibiotic resistance genes are the globally recognized emerging contaminant posing a threat to human health.Intensive livestock is believed to be the major contributor in dissemination of ARGs while antibiotics and metals residues are providing the selective pressure to select for ARGs in the environment.We also performed 16S rRNA microbial community analysis,to gain insights into the microbial population influencing the abundance and diversity of antibiotic resistance genes.For microbial community structure and composition,we amplified the V4-V5 region and the UCHIME algorithm was used to obtain clean reads which were analyzed further by QIIME(Version1.7.0).The bacterial community abundance of 16S rRNA of genomic DNA ranged from 4.22x106 to 2.16x108 gene copy number per gram and 5627 OTUs which were assigned to Bacteria(99.92%),Archaea(0.07%)and others(0.0002%).Proteobacteria were found to be significantly dominant in both soil and manure while Cyanobacteria were significant in feed.Firmicutes showed significant abundance in manure samples.This study was conducted to examine this relationship in a poultry farm environment,to compare the co-occurrence of antibiotics and heavy metal residues with antibiotic resistance genes,we accessed the type and concentration of ARGs in three commercial poultry farms.4.We quantified 284 ARGs,12 mobile genetic elements(MGEs),49 antibiotics residues,7 heavy metals and sequenced the 16S rRNA gene.The abundance and species of ARGs was significantly enriched in manure while soil had the most diverse bacterial composition.Procrustes analysis indicated that ARGs and MGEs at least partly originate from bacteria based on significant correlations between the abundance of ARGs/MGEs and the microbial composition.Cd,As,Zn,Cu and Pb were responsible for a majority of positive correlations with ARGs and antibiotics not being significantly co-related to ARGs compared to metals.Integrons and transposons co-occurred with ARGs corresponding to 9 classes of antibiotics,especially Class1 intergrase intI-1LC.Redundancy analysis(RDA)and Variance partitioning analysis showed that antibiotics,metals,MGEs and bacteria can explain solely 0.7%,5.7%,12.4%,and 21.9%of variances of ARGs community,respectively.These results suggested that bacterial composition and horizontal gene transfer were the major influencing factors on the composition of ARGs;Metals had a bigger effect on ARGs profile than detected antibiotics in this study.5.The highly heavy metal resistant strain Cupriavidus metallidurans BS1 was isolated from the Zijin gold-copper mine in China.This was of particular interest since the extensively studied,closely related strain,C.metallidurans CH34 was shown to not only be highly heavy metal resistant but also able to reduce metal complexes and biomineralizing them into metallic nanoparticles including gold nanoparticles.After isolation,C.metallidurans BS1 was characterized and the complete genome sequenced using PacBio and compared to C.metallidurans CH34.Many heavy metal resistance determinants were identified and shown to have wide-ranging similarities to those of CH34.However,both BS1 and CH34 displayed extensive genome plasticity,probably responsible for significant differences between those strains.BS1 was shown to contain three prophages,not present in CH34,that appear intact and might be responsible for shifting major heavy metal resistance determinants from mega-plasmid to chromid(CHR2)in C.metallidurans BS1.Surprisingly,the single mega-plasmid-pBS1(364.4 kbp)of BS1 contains only a single heavy metal resistance determinant,the czc determinant encoding an RND-type efflux system conferring resistance to cobalt,zinc and cadmium,shown here to be highly similar to the czc determinant located on pMOL30 in C.metallidurans CH34.However,in BS1 another homologous czc determinant was identified on the chromid,most similar to the czc determinant from pMOL30 in CH34.Other heavy metal resistance determinants such as cnr and chr determinants,located on megaplasmid pMOL28 in CH34,were shown to be adjacent to the czc determinant on the chromid(CHR2)in BS1.Additionally,other heavy metal resistance determinants such as pbr,cop,sil,and ars were located on the chromid(CHR2)and not on pBSl in BS1.A diverse range of genomic rearrangements occurred in this strain,isolated from a habitat with constant exposure to high concentrations of copper,gold and other heavy metals.In contrast,the megaplasmid in BS1 contains mostly genes encoding unknown functions,thus might be more of an evolutionary playground where useful genes could be acquired by horizontal gene transfer and possibly reshuffled to help C.metallidurans BS1 withstand the intense pressure of extreme concentrations of heavy metals in its environment.