Diversity Of Tetracycline Resistance Genes Research Articles

High Frequency and Diversity of Tetracycline Resistance Genes in the Microbiota of Broiler Chickens in Tunisia.

Simple SummaryThe extensive use of tetracyclines in clinical practice and livestock has subjected bacterial populations to selection pressure and increased the prevalence of tetracycline resistance, one of the most abundant antibiotic resistances among pathogenic and commensal microorganisms. In the present survey, DNA extracted from cloacal swabs from 195 broiler chickens in Tunisia were molecularly tested for 14 tetracycline resistance genes. A high frequency and diversity of tetracycline resistance genes in the chickens sampled were detected. The results confirm the antimicrobial resistance urgency in Tunisia’s poultry sector and suggest that the investigation of antibiotic resistance genes directly in biological samples could be a useful means for epidemiological studies on the spread of the antimicrobial resistance.Tetracycline resistance is still considered one of the most abundant antibiotic resistances among pathogenic and commensal microorganisms. The aim of this study was to evaluate the prevalence of tetracycline resistance (tet) genes in broiler chickens in Tunisia, and this was done by PCR. Individual cloacal swabs from 195 broiler chickens were collected at two slaughterhouses in the governorate of Ben Arous (Grand Tunis, Tunisia). Chickens were from 7 farms and belonged to 13 lots consisting of 15 animals randomly selected. DNA was extracted and tested for 14 tet genes. All the lots examined were positive for at least 9 tet genes, with an average number of 11 tet genes per lot. Of the 195 animals tested, 194 (99%) were positive for one or more tet genes. Tet(L), tet(M) and tet(O) genes were found in 98% of the samples, followed by tet(A) in 90.2%, tet(K) in 88.7% and tet(Q) in 80%. These results confirm the antimicrobial resistance impact in the Tunisian poultry sector and suggest the urgent need to establish a robust national antimicrobial resistance monitoring plan. Furthermore, the molecular detection of antibiotic resistance genes directly in biological samples seems to be a useful means for epidemiological investigations of the spread of resistance determinants.

Comprehensive screening of genomic and metagenomic data reveals a large diversity of tetracycline resistance genes.

Tetracyclines are broad-spectrum antibiotics used to prevent or treat a variety of bacterial infections. Resistance is often mediated through mobile resistance genes, which encode one of the three main mechanisms: active efflux, ribosomal target protection or enzymatic degradation. In the last few decades, a large number of new tetracycline-resistance genes have been discovered in clinical settings. These genes are hypothesized to originate from environmental and commensal bacteria, but the diversity of tetracycline-resistance determinants that have not yet been mobilized into pathogens is unknown. In this study, we aimed to characterize the potential tetracycline resistome by screening genomic and metagenomic data for novel resistance genes. By using probabilistic models, we predicted 1254 unique putative tetracycline resistance genes, representing 195 gene families (<70 % amino acid sequence identity), whereof 164 families had not been described previously. Out of 17 predicted genes selected for experimental verification, 7 induced a resistance phenotype in an Escherichia coli host. Several of the predicted genes were located on mobile genetic elements or in regions that indicated mobility, suggesting that they easily can be shared between bacteria. Furthermore, phylogenetic analysis indicated several events of horizontal gene transfer between bacterial phyla. Our results also suggested that acquired efflux pumps originate from proteobacterial species, while ribosomal protection genes have been mobilized from Firmicutes and Actinobacteria . This study significantly expands the knowledge of known and putatively novel tetracycline resistance genes, their mobility and evolutionary history. The study also provides insights into the unknown resistome and genes that may be encountered in clinical settings in the future.

Evolution of corresponding resistance genes in the water of fish tanks with multiple stresses of antibiotics and heavy metals

Abuse of antibiotics and heavy metals in aquaculture has been widely concerned and might aggravate the spread of resistance genes in environment. To investigate the occurrence and proliferation of antibiotic resistance genes (ARGs) and heavy metal resistance genes (HMRGs), three commonly used antibiotics (tetracycline, sulfanilamide, cefotaxime) and two heavy metals (Zn and Cu) were designed to add individually or jointly in nine fish tanks including five individual exposure tanks of tetracycline (tet), sulfanilamide (sul), cefotaxime (cef), Cu, Zn and four combination exposure tanks of tetracycline + sulfanilamide (tet + sul), tetracycline + sulfanilamide + cefotaxime (tet + sul + cef), tetracycline + sulfanilamide + Cu (tet + sul + Cu), tetracycline + sulfanilamide + Zn (tet + sul + Zn) as well as the control during the experiment period of 180 days. Nineteen ARGs (tetA, tetB, tetC, tetD, tetE, tetG, tetM, tetO, tetQ, tetS, tetW, tetX, tetY, sul1, sul2, sul3, blaDHA, blaMOX, blaFOX), two HMRGs (copA, czcA) and the class 1 integron gene (intI 1) in fish tanks water were investigated. The results showed that the residual rate of antibiotics and heavy metals ranged from 0.03% to 2.46% and 9.25%–52.97%, respectively, positively related to their original concentration and types. Tetracycline resistance genes were more sensitive to antibiotics and easier to be induced and developed than sulfanilamide resistance genes and AmpC β-lactamase resistance genes. The total relative abundances of ARGs in combined stresses exposure tanks (tet + sul, tet + sul + cef, tet + sul + Cu, tet + sul + Zn) were about 1.01–1.55 times more than the sum of their individual ones. The co-selective effects of cefotaxime on the abundance and diversity of tetracycline resistance genes were stronger than Zn and Cu. Besides, multivariate correlation analysis revealed that tetO, tetQ, tetW and sul3 were in significant correlation with the concentrations of Cu and Zn (r = 0.882–0.992, p < 0.05 or p < 0.01). The significant correlations between tetO and intI1 (p < 0.01), tetW and intI1 (p < 0.05), and sul3 and intI1 (p < 0.05) hinted a potentially serious and undesirable dissemination risk of ribosomal protection proteins gene of ARGs.

Occurrence and diversity of tetracycline resistance genes in the agricultural soils of South Korea

Reports on the occurrence and diversity of antibiotic-resistant bacteria and genes, which are considered to be emerging pollutants worldwide, have, to date, not been published on South Korean agricultural soils. This is the first study to investigate the persistence of tetracycline (oxytetracycline, tetracycline, and chlortetracycline)-resistant bacterial community and genes in natural and long-term fertilized (NPK, pig, and cattle manure composts) agricultural soils in South Korea. The results showed that oxytetracycline and chlortetracycline could be the dominant residues in animal manures; regular fertilization of manures, particularly pig manures, may be the prime cause for the spread and abundance of tetracycline resistance in South Korean agricultural soils. Both the country's natural and agricultural soils are reservoirs of antibiotic-resistant species. Of the 113 tetracycline-resistant isolates identified (19 typical bacterial genera and 36 distinct species), approximately 40 to 99% belonged to Gram-positive bacteria and Bacillus constituted the predominant genera. Of the 24 tet genes targeted, tetG, tetH, tetK, tetY, tetO, tetS, tetW, and tetQ were detected in all soil samples, highlighting their predominance and robust adaptability in soils. Meanwhile, it is suggested that tetC, tetE, tetZ, tetM, tetT, and tetP(B) are the common residues in pig manures, and furthermore, the treatment of soils with pig manures may wield a different impact on the tet gene resistome in agricultural soils. This study thus highlights the necessity for regulating the usage of tetracyclines in South Korean animal farming. This must be followed by proper monitoring of the subsequent usage of animal manures especially that derived from pig farms located in agricultural soils.

Long-term application of fresh and composted manure increase tetracycline resistance in the arable soil of eastern China

The aim of this study was to compare the occurrence, abundance, and diversity of tetracycline resistance genes (tet) in agricultural soils after 6years' application of fresh or composted swine manure. Soil samples were collected from fresh or composted manure-treated farmland at three depths (0–5cm, 5–10cm, and 10–20cm). Nine classes of tet genes [tetW, tetB(P), tetO, tetS, tetC, tetG, tetZ, tetL, and tetX] were detected; tetG, tetZ, tetL, and tetB(P) were predominant in the manure-treated soil. The abundances of tetB(P), tetW, tetC, and tetO were reduced, while tetG and tetL were increased by fertilizing with composted versus fresh manure; thus, the total abundance of tet genes was not significantly reduced by compost manuring. tetG was the most abundant gene in manure-treated soil; the predominant tetG genotypes shared high homology with pathogenic bacteria. The tetG isolates were more diverse in soils treated with fresh versus composted manure, although the residual tet genes in composted manure remain a pollutant and produce a different influence on the tet gene resistome in field soil.

Occurrence, Abundance, and Diversity of Tetracycline Resistance Genes in 15 Sewage Treatment Plants across China and Other Global Locations

Activated sludge was sampled from 15 sewage treatment plants (STPs) across China and other global locations to investigate the occurrence, abundance and diversity of tetracycline resistance genes (tet) in the STPs. Occurrence and abundance of 14 tet genes were determined using polymerase chain reaction (PCR) and quantitative real time PCR. Six genes (tet(A), tet(C), tet(G), tet(M), tet(S), and tet(X)) were detected in all the STPs, while no sludge sample contained tet(Q). Total concentration of the 14 genes was significantly different among the STPs and average tet abundance of the STPs varied greatly among the tet types (p<0.05). Tet(G) had the highest concentration in the STPs, followed by tet(C), tet(A) and tet(S). Phylogenetic diversity of the genes was investigated using DNA cloning. BLAST analysis showed that all of the 450 cloned sequences matched known tet genes, except for tet(G). The 56 tet(G) clones were grouped into 14 genotypes, among which type G24 had an identical sequence to tet(G) carried by Salmonella enterica or Acinetobacter baumannii, while the other sequences had low similarity to the known genes in GenBank. The results of this study might be useful to understand the diversity of these resistance genes in STPs.

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.

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.

Diversity of tetracycline resistance genes in bacteria from aquaculture sources in Australia

To determine the genetic determinants responsible for tetracycline resistance in oxytetracycline resistant bacteria from aquaculture sources in Australia. Twenty of 104 (19%) isolates tested were resistant to oxytetracycline (MIC > or = 16 microg ml(-1)). Using polymerase chain reaction (PCR) amplification, one or more tet genes were detected in 15/20 (75%) isolates tested, but none were found in 5/20 (25%). tetM (50%) was the most common determinant, followed by tetE (45%), tetA (35%) and tetD (15%). Five of 12 oxytetracycline resistant isolates studied were able to transfer their R-plasmid to Escherichia coli recipients of chicken, pig and human origin. tetA, tetD and tetM were found to be transferred while tetE was not transferred. Southern hybridization and PCR were used to confirm transfer of determinants. Bacterial isolates from aquaculture sources in Australia harbour a variety of tetracycline resistance genes, which can be transferred to other bacteria of different origin. Bacteria from aquaculture sources in Australia contribute to the resistance gene pool reservoir. The in vitro transfer of tetracycline R-plasmid from aquatic bacteria to E. coli isolates from various sources is an indication of the potential public health risk associated with these resistance determinants.

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.

Diversity of Tetracycline Resistance Genes in Bacteria Isolated from Various Agricultural Environments

A total of 350 tetracycline-resistant (Tcr) bacteria were isolated from livestock feces, farmyard manure (FYM), and soil in Japan. The isolates were classified into 28 established genera of the phyla Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria by studying partial 16S rRNA gene sequences. PCR analysis revealed that 249 of the 350 Tcr isolates contained at least 1 of 15 Tcr genes (tet genes): 140 isolates contained an efflux pump gene and 109 isolates contained a ribosomal protection protein (RPP) gene. The predominant fecal isolates were identified as members of the genera Enterococcus and Escherichia, carrying tet (M) and tet (H), respectively. The predominant soil isolates were identified as members of the genera Burkholderia and Afipia, carrying tet (Z) and tet (B), respectively. This study is the first to describe the presence of tet genes in members of the genera Bordetella, Bradyrhizobium, Burkholderia, Dyella, Flexibacter, Kurthia, Luteibacter, Lysobacter, "Nordella", Ochrobactrum, Pediococcus, Rhodopseudomonas, and Vagococcus. It has revealed that tet genes are distributed in much more diverse phylogenetic groups of bacteria in the environment than has been reported previously.

Diversity of tetracycline resistance genes in bacteria from Chilean salmon farms.

Twenty-five distinct tetracycline-resistant gram-negative bacteria recovered from four Chilean fish farms with no history of recent antibiotic use were examined for the presence of tetracycline resistance (tet) genes. Sixty percent of the isolates carried 1 of the 22 known tet genes examined. The distribution was as follows. The tet(A) gene was found in six isolates. The tet(B) gene was found in two isolates, including the first description in the genus Brevundimonas: Two isolates carried the tet(34) and tet(B) genes, including the first description of the tet(34) gene in Pseudomonas and Serratia and the first description of the tet(B) gene in Pseudomonas: The tet(H) gene was found in two isolates, which includes the first description in the genera Moraxella and Acinetobacter: One isolate carried tet(E), and one isolate carried tet(35), the first description of the gene in the genus Stenotrophomonas: Finally, one isolate carried tet(L), found for the first time in the genus Morganella: By DNA sequence analysis, the two tet(H) genes were indistinguishable from the previously sequenced tet(H) gene from Tn5706 found in Pasteurella multocida. The Acinetobacter radioresistens isolate also harbored the Tn5706-associated 1,063-bp IS element IS1597, while the Moraxella isolate carried a 1,026-bp IS-like element whose 293-amino-acid transposase protein exhibited 69% identity and 84% similarity to the transposase protein of IS1597, suggesting the presence of a novel IS element. The distribution of tet genes from the Chilean freshwater ponds was different than those that have previously been described from other geographical locations, with 40% of the isolates carrying unidentified tetracycline resistance genes.

Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities.

In this study, we used PCR typing methods to assess the presence of tetracycline resistance determinants conferring ribosomal protection in waste lagoons and in groundwater underlying two swine farms. All eight classes of genes encoding this mechanism of resistance [tet(O), tet(Q), tet(W), tet(M), tetB(P), tet(S), tet(T), and otrA] were found in total DNA extracted from water of two lagoons. These determinants were found to be seeping into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. The identities and origin of these genes in groundwater were confirmed by PCR-denaturing gradient gel electrophoresis and sequence analyses. Tetracycline-resistant bacterial isolates from groundwater harbored the tet(M) gene, which was not predominant in the environmental samples and was identical to tet(M) from the lagoons. The presence of this gene in some typical soil inhabitants suggests that the vector of antibiotic resistance gene dissemination is not limited to strains of gastrointestinal origin carrying the gene but can be mobilized into the indigenous soil microbiota. This study demonstrated that tet genes occur in the environment as a direct result of agriculture and suggested that groundwater may be a potential source of antibiotic resistance in the food chain.