Ribosomal Protection Protein Genes Research Articles

Tetracycline-resistant bacteria and ribosomal protection protein genes in soils from selected agricultural fields and livestock farms

Antibiotic resistance in soil environment has eminently been compared and studied between agricultural and pristine soils, and the role of concentrated animal feeding operations has markedly been recognized as one of the major sources of antibiotic resistance. This study described the tetracycline resistance in small-scale farms in pursuit of presenting its possible role and contribution to the persistence of antibiotic resistance in the environment. Results of the study would render additional information on the occurrence of the ribosomal protection protein (RPP) tet genes among the isolated bacteria from the selected agricultural soils. Four tetracycline resistance and RPP genes were determined in two different agricultural soil settings. Both the culture and molecular method were used to determine and measure tetracycline resistance in soils from arable land and animal house. Results revealed a significantly higher number of culturable antibiotic-resistant bacteria in animal houses than arable lands which was suggestive of higher antibiotic resistance in areas where there was direct administration of the antibiotics. However, quantification of the gene copy numbers in the agricultural soils indicated a different result. Higher gene copy number of tetO was determined in one animal house (IAH-3), while the two other tet genes tetQ and tetW were found to be higher in arable lands. Of the total 110 bacterial isolates, tetW gene was frequently detected, while tetO gene was absent in any of the culturable bacterial isolates. Principal component analysis of occurrence and gene copy number of RPP tet genes tetO, tetQ, and tetW also revealed highest abundance of RPP tet genes in the manure and arable soils. Another important highlight of this study was the similarity of the RPP tet genes detected in the isolated bacteria from the agricultural soils to the identified RPP tet genes among pathogenic bacteria. Some of the tetracycline-resistant bacterial isolates were also multidrug resistant as it displayed resistance to tetracycline, erythromycin, and streptomycin using disk diffusion testing.

Antibiotic resistance gene abundances associated with heavy metals and antibiotics in the sediments of Changshou Lake in the three Gorges Reservoir area, China

• Long-time aquaculture have an impact on the antibiotic pollution in sediment. • Sulfamethoxazole and tetracycline posed relatively high ecological risks. • There are significant correlations between different tetracycline ARGs. • Significant correlations were observed between heavy metals and antibiotics/ARGs. Human activities have very important influence on the migration and transformation of antibiotics and antibiotic resistance genes (ARGs). In the present study, Changshou Lake, located in the Three Gorges Reservoir area, was selected to investigate the abundance of heavy metals, antibiotics and ARGs. In addition, the environmental risks of the antibiotics were evaluated and the relationships between heavy metals and antibiotics/ARGs were assessed. Higher concentrations of antibiotics and ARGs were observed in the ecological aquaculture area of the Changshou Lake. The results suggested that the ecological risk of tetracycline and sulfamethoxazole was relatively higher than that of other antibiotics. The results of the cumulative risk quotient showed that the target antibiotics of the ecological aquaculture area (sites C1 and C3) seriously threaten the ecological environment of Changshou Lake, and antibiotic pollution in the sediment environment cannot be ignored. Especially for the tetracycline ARGs, there was a positive relationship among efflux pump genes ( tetA , tetC and tetG ) at the P < 0.05 level, and there was also a strong correlation between the ribosomal protection protein genes, except tetQ . The joint pollution of heavy metals and antibiotics in Changshou Lake may be due to the cage aquaculture in the early stage of the lake. In addition, positive significant correlations were observed between ARGs/antibiotics and heavy metals in Changshou Lake, highlighting the potential pressures of heavy metals on the ARGs/antibiotics.

Increased occurrence of heavy metals, antibiotics and resistance genes in surface soil after long-term application of manure

The purpose of this study was to investigate the impact of long-term application of pig manure on the accumulation of heavy metals, antibiotics and ARGs in surface soil sampled from the Jiaxing long-term field experimental site with three manure treatments, N-PM (0 kg/ha/y, dw), L-PM (7720 kg/ha/y, dw), and H-PM (11,580 kg/ha/y, dw), in 2013 and 2014. The results showed that most serious metal pollution of Zn and Cu was recorded in all manured samples in both years, and their contents exceeded the soil quality standards. Among the three tetracyclines, chlortetracycline was the predominant antibiotic detected with a range of 3.04–98.03 μg·kg−1 in 2013 and 28.67–344.74 μg·kg−1 in 2014 after long-term pig manure application. Q-PCR results showed that the average accumulation of ribosomal protection protein genes (tetM, tetO, tetQ and tetW) was lower than most of the efflux pump genes (tetA and tetG). The abundance of tet and sul genes of those sites with manure application was significantly higher than that of sites without manure application in both years. Metagenomics analysis of ARGs revealed that the abundance of multidrug resistance genes was the most abundant subtype, followed by fluoroquinolone, bacitracin, sulfonamide and tetracycline. There was a positive correlation between the levels of ARGs; soil organic matter, antibiotics, Cu, As, and Zn levels in both years. These results may shed light on the mechanism underlining the effects of long-term manure application on the occurrence and dissemination of ARGs in surface soil.

No evidential correlation between veterinary antibiotic degradation ability and resistance genes in microorganisms during the biodegradation of doxycycline

Biodegradation of antibiotic residues in the environment by microorganisms may lead to the generation of antibiotic resistance genes (ARGs), which are of great concern to human health. The aim of this study was to determine whether there is a relationship between the ability to degrade antibiotic doxycycline (DOX) and the development of resistance genes in microorganisms. We isolated and identified ten bacterial strains from a vegetable field that had received long-term manure application as fertilizer and were capable of surviving in a series of DOX concentrations (25, 50, 80, and 100mg/L). Our results showed no evidential correlation between DOX degradation ability and the development of resistance genes among the isolated microorganisms that had high DOX degradation capability (P > 0.05). This was based on the fact that Escherichia sp. and Candida sp. were the most efficient bacterial strains to degrade DOX (92.52% and 91.63%, respectively), but their tetracycline resistance genes showed a relatively low risk of antibiotic resistance in a 7-day experiment. Moreover, the tetM of the ribosomal protection protein genes carried by these two preponderant bacteria was five-fold higher than that carried by other isolates (P < 0.05). Pearson correlations between the Ct/C0 of DOX and tet resistance genes of three isolates, except for Escherichia sp. and Candida sp., showed remarkable negative correlations (P < 0.05), mainly because tetG markedly increased during the DOX degradation process. Our results concluded that the biodegradation of antibiotic residues may not necessarily lead to the development of ARGs in the environment. In addition, the two bacteria that we isolated, namely, Escherichia sp. and Candida sp., are potential candidates for the engineering of environmentally friendly bacteria.

Antibiotic resistance genes in surface water of eutrophic urban lakes are related to heavy metals, antibiotics, lake morphology and anthropic impact.

Urban lakes are impacted by heavy human activities and represent potential reservoirs for antibiotic resistance genes. In this study, six urban lakes in Wuhan, central China were selected to analyze the distribution of sulfonamide resistance (sul) genes, tetracycline resistance (tet) genes and quinolone resistance (qnr) genes and their relationship with heavy metals, antibiotics, lake morphology and anthropic impact. sul1 and sul2 were detected in all six lakes and dominated the types of antibiotic resistance genes, which accounted for 86.28-97.79% of the total antibiotic resistance gene abundance. For eight tested tet genes, antibiotic efflux pumps (tetA, tetB, tetC, and tetG) genes were all observed in six lakes and had higher relative abundance than ribosomal protection protein genes (tetM and tetQ). For 4 plasmid mediated quinolone resistance genes, only qnrD is found in all six lakes. The class I integron (intI1) is also found to be a very important media for antibiotic resistance gene propagation in urban lakes. The results of redundancy analysis and variation partitioning analysis showed that antibiotic and co-selection with heavy metals were the major factors driving the propagation of antibiotic resistance genes in six urban lakes. The heavily eutrophic Nanhu Lake and Shahu Lake which located in a high density building area with heavy human activities had the higher relative abundance of total antibiotic resistance genes. Our study could provide a useful reference for antibiotic resistance gene abundance in urban lakes with high anthropic impact.

Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.

Safety Evaluation Of Sjenica Cheese With Regard To Coagulase-Positive Staphylococci And Antibiotic Resistance Of Lactic Acid Bacteria And Staphylococci

Abstract Sjenica cheese is an artisanal cheese stored in brine, traditionally produced from raw sheep’s milk in the southern part of Serbia - Sjenica Pester plateau. The aim of this study was to perform the safety evaluation of Sjenica cheese. As one of the safety criteria we considered the number of coagulase positive staphylococci and their enterotoxigenic potential. Antibiotic susceptibility/resistance patterns of autochthonous lactic acid bacteria and coagulase-positive staphylococci isolated from Sjenica cheese was also investigated. During the monitoring period of the cheese-making process, coagulase positive staphylococci did not reach the value of 105 cfu/g. Among coagulase positive staphylococci, 12 (46,15%) isolates showed enterotoxigenic potential and were identified as Staphylococcus intermedius (11 isolates) and Staphylococcus aureus (1 isolate). Vancomycin resistance was the most prevalent phenotypic resistance profile in coagulase positive staphylococci. Lactococci present the most dominant population among lactic acid bacteria. The most prevalent resistance phenotype in lactococci was resistance to streptomycin (83.33%), ampicillin and penicillin (70.83%); lactobacilli were characterized by resistance to vancomycin (62.5%) and tetracycline (54.17%), while resistance to streptomycin (82.46%) was the most prevalent phenotypic profile in enterococci. All coagulase positive staphylococci and lactic acid bacteria isolates that showed resistance to tetracycline on disc diffusion and E-test, were tested for the presence of ribosomal protection proteins, tet(M) and tet(K) genes. All isolates were positive for ribosomal protection proteins genes; 14 (60.87%) isolates showed tet(M) gene presence, while 2 lactobacilli isolates revealed the presence of tet(K) gene.

Behavior of antibiotics and antibiotic resistance genes in eco-agricultural system: A case study

This study aims to determine abundance and persistence of antibiotics and antibiotic resistance genes (ARGs) in eco-agricultural system (EAS), which starts from swine feces to anaerobic digestion products, then application of anaerobic digestion solid residue (ADSR) and anaerobic digestion liquid residue (ADLR) to the soil to grow ryegrass, one of swine feed. Oxytetracycline had the highest concentration in manure reaching up to 138.7mg/kg. Most of antibiotics could be effectively eliminated by anaerobic digestion and removal rates ranged from 11% to 86%. ARGs abundance fluctuated within EAS. TetQ had the highest relative abundance and the relative abundance of tetG had the least variation within the system, which indicates that tetG is persistent in the agricultural environment and requires more attention. Compared to the relative abundance in manure, tetC and tetM increased in biogas residue while three ribosomal protection proteins genes (tetO, tetQ, tetW) decreased (p<0.05), with other genes showing no significant change after anaerobic fermentation (p>0.05). Most ARGs in downstream components (soils and fishpond) of EAS showed significantly higher relative abundance than the control agricultural system (p<0.05), except for tetG and sulI.

Fate of trace tetracycline with resistant bacteria and resistance genes in an improved AAO wastewater treatment plant

The fate of trace tetracycline, tetracycline resistant bacteria (TRB) and tetracycline resistant genes (TRGs) in an improved anaerobic-anoxic-oxic (AAO) wastewater treatment plant (WWTP) was investigated in this study. Quantitative real-time polymerase chain reaction (qPCR) and conventional heterotrophic plate count method were used to measure eight tet genes (tetA, tetB, tetC, tetE, tetM, tetO, tetS and tetX) and TRB, respectively. The TRB percent of total heterotrophic bacteria (THB) is about 1.31–24.1% in WWTP influent. Tet gene abundance in the WWTP varied greatly among the gene types. The concentrations of TRGs in effluent samples ranged from 7.11×10−9 to 1.53×10−4 copies/copy 16S rRNA gene. TRB and THB, tetM and tetO, tetE and tetX, but not the others, showed a significant correlation with each other (p<0.01). The relationships between ribosomal protection protein genes, enzymatic modification gene and corresponding concentrations of antibiotics were found to be considerably significant (R2=0.898, p<0.01 for ribosomal protection protein genes and R2=0.872, p<0.05 for enzymatic modification gene).

Abundance and persistence of antibiotic resistance genes in livestock farms: A comprehensive investigation in eastern China

Increases of antibiotic resistance genes in the environment may pose a threat to public health. The purpose of this study was to investigate the abundance and diversity of tetracycline (tet) and sulfonamide (sul) resistance genes in eight livestock farms in Hangzhou, eastern China. Ten tet genes (tetA, tetB, tetC, tetG, tetL, tetM, tetO, tetQ, tetW, and tetX), two sul genes (sulI and sulII), and one genetic element associated with mobile antibiotic resistance genes [class 1 integron (intI1)] were quantified by real-time polymerase chain reaction. No significant difference was found in the abundance of the tet and sul genes in various scales of pig, chicken, and duck farms (P>0.05). The average abundance of ribosomal protection protein genes (tetQ, tetM, tetW, and tetO) in the manure and wastewater samples was higher than most of the efflux pump genes (tetA, tetB, tetC, and tetL) and enzymatic modification gene (tetX) (P<0.05), except for efflux pump gene tetG, which was abundant and showed no difference from tetM. Most ARGs had higher relative abundance in the wastewater lagoon than in manures even after treatment. Although the three ribosomal protection protein genes (tetQ, tetW, and tetO) had higher relative abundance, numbers were reduced during the complete wastewater treatment process in pig farms (P<0.05). The relative abundance of tetG, sulI, and sulII increased after the wastewater treatment and the removal of these three genes exhibited significant positive correlations with the intI1 gene (tetG: R2=0.60, P<0.05; sulI: R2=0.72, P<0.05; sulII: R2=0.62, P<0.05), suggesting that intI1 may be involved in their proliferation. As for tetM and sulII genes, a highly significant difference was found in manure samples between pig farms and duck farms (P<0.001). Phylogenetic analysis showed that tetM was more diverse in duck farms than in pig farms. Additionally, sulII sequence was conserved both in pig and duck farms. This is the first comprehensive study to detail the relative abundance of specific ARGs in animal manures and agricultural wastewater treatment systems, potentially providing knowledge for managing antibiotic resistance emanating from agricultural activities.

Human pathogens and tetracycline-resistant bacteria in bioaerosols of swine confinement buildings and in nasal flora of hog producers

Swine confinement buildings in eastern Canada are enclosed and equipped with modern production systems to manage waste. Bioaerosols of these swine confinement buildings could be contaminated by human pathogens and antimicrobial resistant bacteria which could colonize exposed workers. We therefore wanted to analyze bioaerosols of swine confinement buildings and nasal flora of Canadian hog producers to evaluate possible colonization with human pathogens and tetracycline-resistant bacteria. Culturable and non-culturable human pathogens and tet genes were investigated in the bioaerosols of 18 barns. The nasal passages of 35 hog producers were sampled and total DNA was extracted from the calcium-alginate swabs to detect, by PCR, Campylobacter, C. perfringens, Enterococcus, E. coli, Y. enterocolitica, tetA/tetC, tetG and ribosomal protection protein genes. Airborne culturable C. perfringens, Enterococcus, E. coli, and Y. enterocolitica were present in the bioaerosols of 16, 17, 11 and 6 of the 18 facilities. Aerosolized total (culturable/non culturable) Campylobacter, C. perfringens, Enterococcus, E. coli and Y. enterocolitica were detected in 10, 6, 15, 18 and 2 barns, respectively. Tet genes were found in isolates of culturable human pathogens. TetA/tetC, tetG and ribosomal protection protein genes were detected in the bioaerosols of all 18 studied buildings. Campylobacter, C. perfringens, Enterococcus, E. coli, and Y. enterocolitica were found respectively in 4, 9, 17, 14 and one nasal flora of workers. One and 10 workers were positive for tetA/tetC and tetG genes, respectively. In swine confinement buildings, hog producers are exposed to aerosolized human pathogens and tetracycline-resistant bacteria that can contaminate the nasal flora.

Abundance and Diversity of Tetracycline Resistance Genes in Soils Adjacent to Representative Swine Feedlots in China

Tetracyclines are commonly used antibiotics in the swine industry for disease treatment and growth promotion. Tetracycline resistance was determined in soils sampled from farmlands in the vicinity of nine swine farms located in three cities in China. Totally, 15 tetracycline resistance (tet) genes were commonly detected in soil samples, including seven efflux pump genes (tetA, tetC, tetE, tetG, tetK, tetL, tetA/P), seven ribosomal protection proteins (RPPs) genes (tetM, tetO, tetQ, tetS, tetT, tetW, tetB/P), and one enzymatic modification gene (tetX). The quantitative real-time PCR was further used to quantify five RPPs genes (tetM, tetO, tetQ, tetW, tetT) and 16S rRNA gene abundances. The concentrations of total tetracyclines (5 typical tetracyclines and 10 of their degradation products) in these soils were measured using liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) and were found to range from 5.4 to 377.8 μg·kg(-1) dry soil. Bivariate correlation analysis confirmed that absolute tet gene copies (sum of tetM, tetO, tetQ, tetW genes) were strongly correlated with the concentrations of tetracycline residues (r(2) = 0.45, P < 0.05), ambient bacterial 16S-rRNA gene copies in each soil sample (r(2) = 0.65, P < 0.01), and organic matter in soil (r(2) = 0.46, P < 0.05), respectively. Finally, the phylogenetic analysis on tetM combined with culture-independent molecular techniques revealed at least five genotypes of tetM in nine soil samples.

High Occurrence Rate of Tetracycline (TC)-Resistant Bacteria and TC Resistance Genes Relates to Microbial Diversity in Sediment of Mekong River Main Waterway

Spatial monitoring of tetracycline (TC)-resistant bacteria in sediments of the Mekong River watershed revealed that the main waterway showed a high occurrence rate of TC-resistant bacteria, whereas Tonle Sap Lake and the Sai Gon estuary did not. The Shannon index (H'), an indicator of ecological diversity, was calculated from denaturing gradient gel electrophoresis (DGGE) profiles, which indicated that the main waterway of the Mekong River had high microbial diversity (high H') compared to Tonle Sap Lake and the Sai Gon estuary; this diversity was positively correlated with the occurrence rate of TC-resistant bacteria. Analysis of ribosomal protection protein (RPP) genes tet(M), tet(S) and tet(W) in the same area also revealed that high diversity was positively correlated with the occurrence rate of RPP genes, suggesting that RPP genes are well conserved across various bacterial species. Further evidence of different genotypes of tet(M) suggests that the drug resistance genes likely have various origins, and are mixed in the sediment. Sediments in this area are therefore potential reservoirs of drug resistance genes.

Diversity of tet resistance genes in tetracycline-resistant bacteria isolated from a swine lagoon with low antibiotic impact

Tetracycline resistance has been extensively studied and shown to be widespread. A number of previous studies have clearly demonstrated that a variety of tetracycline resistance genes are present in swine fecal material, treatment lagoons, and the environments surrounding concentrated animal feeding operations (CAFOs). The diversity of tetracycline resistance within a swine lagoon located at a CAFO that used only bacitricin methylene disalicylate as an antibiotic was evaluated by screening 85 tetracycline-resistant isolates for the presence of 18 different genes by performing PCR with primers that target tetracycline efflux genes of Gram-negative bacteria and ribosomal protection proteins. In addition, partial 16S rRNA sequences from each of these isolates were sequenced to determine the identity of these isolates. Of the 85 isolates examined, 17 may represent potential novel species based on BLAST results. Greater than 50% of the isolates (48 out of 85) were found to not contain targeted tet efflux genes. Though minimum inhibitory concentrations ranged widely (16 - >256 mg/L), these values did not give an indication of the tet genes present. Ten new genera were identified that contain at least one tet efflux gene. Five other genera possessed tet efflux genes that were not found in these organisms previously. Interestingly, none of the isolates possessed any of the selected ribosomal protection protein genes. Though tetracycline resistance was found in bacteria isolated from a swine CAFO lagoon, it appears that the limited antibiotic use at this CAFO might have impacted the presence and diversity of tetracycline resistance genes.

Distribution and diversity of tetracycline resistance genes encoding ribosomal protection proteins in Mekong river sediments in Vietnam

We investigated the distribution and diversity of tetracycline resistance genes encoding ribosomal protection proteins (RPPs) in river and channel sediments of the Mekong Delta in Vietnam. The sediment samples were taken from nine sites in the Hau River in southern Vietnam and from 1 site in a channel in Can Tho City in May 2004 using an Ekman-Birge sediment surface sampler. The RPP genes were amplified using PCR with DNA templates obtained directly from the sediments. The tet(M), tet(S), and tet(W) genes were detected by PCR in most sediment samples. Denaturing gradient gel electrophoresis analysis of these genes and sequencing of the resulting bands showed that tet(S) and tet(W) had only one genotype each, but that tet(M) had at least two, which were tentatively called type 1 and type 2. Type 1 tet(M) was identical to the gene encoded in various plasmids and transposons of gram-positive and gram-negative bacteria, and type 2tet(M) was similar to the gene encoded in Tn1545 of Enterococcus faecalis (99% identity, 170 bp/171 bp). This study showed that various RPP genes were widely distributed in the river and channel sediments of the Mekong Delta.