Conferring Tetracycline Resistance Research Articles

Captivity increased the abundance of high-risk antibiotic resistance genes in the giant panda gut microbiome

Captivity is a key strategy for protecting endangered species, but research has primarily focused on artificial breeding and reintroduction to bolster wild populations, often overlooking the environmental and health risks associated with antibiotic resistance genes (ARGs). Here, we conducted a comprehensive analysis of the microbiome and ARG profiles in the gut of wild giant pandas across five representative populations, as well as one captive population, utilizing 16S rRNA gene sequencing and High-Throughput Quantitative PCR. Our findings revealed that both geographic location and captivity significantly influenced the gut microbial community and ARG composition in the gut of giant pandas. Additionally, we identified core microbiomes with essential ecological functions, particularly those related to food utilization, were identified in the giant panda gut across different regions. The gut ARGs in giant pandas exhibited a broad range of subtypes, with multidrug resistance genes being the most prevalent. Notably, the captive population harbored the highest abundance of high-risk ARGs, especially those conferring tetracycline resistance. High-risk multidrug ARGs (e.g., tolC, mepA, and mdtA) were found to be strongly correlated with the potential pathogens, such as Escherichia_Shigellina and Pseudomonas. Furthermore, bamboo-associated ARGs and mobile genetic elements (MGEs) contributed significantly to the ARG abundance in the giant panda gut, indicating that diet plays a crucial role in shaping gut resistome. Collectively, our study provides a detailed mapping of giant panda gut microbiomes and ARG distribution, offering valuable insights for conservation efforts and advancing our understanding of ARG dynamics in giant panda populations.

Detecting horizontal gene transfer with metagenomics co-barcoding sequencing.

Horizontal gene transfer (HGT) is the process through which genetic information is transferred between different genomes and that played a crucial role in bacterial evolution. HGT can enable bacteria to rapidly acquire antibiotic resistance and bacteria that have acquired resistance is spreading within the microbiome. Conventional methods of characterizing HGT patterns include short-read metagenomic sequencing (short-reads mNGS), long-read sequencing, and single-cell sequencing. These approaches present several limitations, such as short-read fragments, high amounts of input DNA, and sequencing costs, respectively. Here, we attempt to circumvent present limitations to detect HGT by developing a metagenomics co-barcode sequencing workflow (MECOS) and applying it to the human and mouse gut microbiomes. In addition to that, we have over 10-fold increased contig length compared to short-reads mNGS; we also obtained exceeding 30 million paired reads with co-barcode information. Applying the novel bioinformatic pipeline, we integrated this co-barcoding information and the context information from long reads, and observed over 50-fold HGT events after we corrected the potential wrong HGT events. Specifically, we detected approximately 3,000 HGT blocks in individual samples, encompassing ~6,000 genes and ~100 taxonomic groups, including loci conferring tetracycline resistance through ribosomal protection. MECOS provides a valuable tool for investigating HGT and advance our understanding on the evolution of natural microbial communities within hosts.IMPORTANCEIn this study, to better identify horizontal gene transfer (HGT) in individual samples, we introduce a new co-barcoding sequencing system called metagenomics co-barcoding sequencing (MECOS), which has three significant improvements: (i) long DNA fragment extraction, (ii) a special transposome insertion, (iii) hybridization of DNA to barcode beads, and (4) an integrated bioinformatic pipeline. Using our approach, we have over 10-fold increased contig length compared to short-reads mNGS, and observed over 50-fold HGT events after we corrected the potential wrong HGT events. Our results indicate the presence of approximately 3,000 HGT blocks, involving roughly 6,000 genes and 100 taxonomic groups in individual samples. Notably, these HGT events are predominantly enriched in genes that confer tetracycline resistance via ribosomal protection. MECOS is a useful tool for investigating HGT and the evolution of natural microbial communities within hosts, thereby advancing our understanding of microbial ecology and evolution.

Food risk associated with vegetable consumption, exposure to antimicrobial-resistant strains and pesticide residues

This preliminary study aimed to detect biological and chemical contaminants in vegetables sold in Sicily for human consumption, assess the spread of antimicrobial-resistant (AMR) strains in these foods, and characterize their antimicrobial-resistance genes. A total of 29 fresh and ready-to-eat samples were analyzed. Microbiological analyses were performed for the detection of Salmonella spp. and the enumeration of Enterococci, Enterobacteriaceae, and Escherichia coli. Antimicrobial resistance was assessed by the Kirby-Bauer method, according to the Clinical and Laboratory Standards Institute guidelines. Pesticides were detected by high-performance liquid chromatography and gas chromatography coupled with mass spectrometry. No samples were contaminated by Salmonella spp., E. coli was detected in 1 sample of fresh lettuce at a low bacterial count (2 log cfu/g). 17.24% of vegetables were contaminated by Enterococci and 65.5% by Enterobacteriaceae (bacterial counts between 1.56 log cfu/g and 5.93 log cfu/g and between 1.6 log cfu/g and 5.48 log cfu/g respectively). From 86.2% of vegetables, 53 AMR strains were isolated, and 10/53 isolates were multidrug resistant. Molecular analysis showed that the blaTEM gene was detected in 12/38 β-lactam-resistant/intermediate-resistant isolates. Genes conferring tetracycline resistance (tetA, tetB, tetC, tetD, tetW) were detected in 7/10 isolates. The qnrS gene was detected in 1/5 quinolone-resistant isolates, the sulI gene was detected in 1/4 sulfonamide-resistant/intermediate-resistant isolates and the sulIII gene was never detected. Pesticides were detected in 27.3% of samples, all of which were leafy vegetables. Despite the satisfactory hygienic status of samples, the high percentage of AMR bacteria detected stresses the need for an effective monitoring of these foods as well as adequate strategies to counteract the spread of AMR bacteria along the agricultural chain. Also, the chemical contamination of vegetables should not be underestimated, especially considering that leafy vegetables are commonly consumed raw and that no official guidelines about maximum residue limits of pesticides in ready-to-eat vegetables are available.

Evolution, persistence, and host adaption of a gonococcal AMR plasmid that emerged in the pre-antibiotic era.

Plasmids are diverse extrachromosomal elements significantly that contribute to interspecies dissemination of antimicrobial resistance (AMR) genes. However, within clinically important bacteria, plasmids can exhibit unexpected narrow host ranges, a phenomenon that has scarcely been examined. Here we show that pConj is largely restricted to the human-specific pathogen, Neisseria gonorrhoeae. pConj can confer tetracycline resistance and is central to the dissemination of other AMR plasmids. We tracked pConj evolution from the pre-antibiotic era 80 years ago to the modern day and demonstrate that, aside from limited gene acquisition and loss events, pConj is remarkably conserved. Notably, pConj has remained prevalent in gonococcal populations despite cessation of tetracycline use, thereby demonstrating pConj adaptation to its host. Equally, pConj imposes no measurable fitness costs and is stably inherited by the gonococcus. Its maintenance depends on the co-operative activity of plasmid-encoded Toxin:Antitoxin (TA) and partitioning systems rather than host factors. An orphan VapD toxin encoded on pConj forms a split TA with antitoxins expressed from an ancestral co-resident plasmid or a horizontally-acquired chromosomal island, potentially explaining pConj's limited distribution. Finally, ciprofloxacin can induce loss of this highly stable plasmid, reflecting epidemiological evidence of transient reduction in pConj prevalence when fluoroquinolones were introduced to treat gonorrhoea.

Molecular Detection of Metronidazole and Tetracycline Resistance Genes in Helicobacter pylori-Like Positive Gastric Samples from Pigs.

Antimicrobial resistance is a major public health concern. The aim of this study was to assess the presence of antibiotic resistance genes, previously reported in Helicobacter pylori, in gastric samples of 36 pigs, in which DNA of H. pylori-like organisms had been detected. Based on PCR and sequencing analysis, two samples were positive for the 16S rRNA mutation gene, conferring tetracycline resistance, and one sample was positive for the frxA gene with a single nucleotide polymorphism, conferring metronidazole resistance. All three amplicons showed the highest homology with H. pylori-associated antibiotic resistance gene sequences. These findings indicate that acquired antimicrobial resistance may occur in H. pylori-like organisms associated with pigs.

Role of Staphylococcus aureus Tet38 in Transport of Tetracycline and Its Regulation in a Salt Stress Environment.

Staphylococcus aureus Tet38 efflux pump has multiple functions, including conferring resistance to tetracycline and other compounds and enabling internalization and survival within epithelial cells. In this study, we evaluated the effects of sodium and potassium on tet38 expression. These monovalent cations are known to play a role in transport by the related S. aureus TetK and B. subtilis TetL transporters. tet38 transcription decreased with increasing sodium concentrations by means of direct repression by the salt stress-dependent KdpD/E regulator. tet38 transcription increased 20-fold and tetracycline minimum inhibitory concentration (MIC) increased 4-fold in a ΔkdpD mutant. KdpE bound specifically to the tet38 promoter. Under extreme salt stress, the survival of S. aureus with intact tet38 was reduced compared to that of a Δtet38 mutant. To study the effect of sodium on Tet38 function, we generated constructs overexpressing tet38 and tetK and introduced them into Escherichia coli TO114, which is deficient in major sodium transporters. Tet38 tetracycline efflux was directly demonstrated in a fluorescence assay, and tetracycline efflux of both Tet38 and TetK was abolished by the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP). In contrast, NaCl inhibited efflux by Tet38 but not TetK, whereas KCl inhibited efflux by TetK but not Tet38. Cell-associated Na increased with heterologous overexpression of Tet38. These data indicate that S. aureus Tet38 is a tetracycline efflux pump regulated by the KdpD/E regulator. Under salt stress, S. aureus adjusted its survival in part by reducing the expression of tet38 through KdpD/E. The mechanisms by which Tet38 is detrimental to salt tolerance in S. aureus and inhibited by sodium remain to be determined. IMPORTANCE This study shows that S. aureus Tet38 is a tetracycline efflux pump regulated by KdpD/E regulator. These findings are the first direct demonstration of Tet38-mediated tetracycline efflux, which had previously been inferred from its ability to confer tetracycline resistance. Under salt stress, S. aureus adjusts its survival in part by reducing the expression of tet38 through KdpD/E. We demonstrated the differences in the respective functions of S. aureus Tet38 and other tetracycline efflux transporters (S. aureus TetK, B. subtilis TetL) regarding their transport of tetracycline and Na+/K+. Notably, sodium selectively reduced tetracycline efflux by Tet38, and potassium selectively reduced tetracycline efflux by TetK. The multiple functions of Tet38 emphasize its importance in bacterial adaptation to and survival in diverse environments.

Small RNA-mediated regulation of the tet(M) resistance gene expression in Enterococcus faecium

We investigated the role of a novel small RNA expressed in Enterococcus faecium (named Ern0030). We revealed that ern0030 was encoded within the 5′untranslated region of tet(M), a gene conferring tetracycline resistance through ribosomal protection. By RACE mapping, we accurately determined the boundaries of ern0030, which corresponded to Ptet. This upstream sequence of tet(M), Ptet, was previously described within transcriptional attenuation mechanism. Here, Northern blot analyses revealed three transcripts of different lengths (ca. 230, 150 and 100 nucleotides) expressed from Ptet. Phenotypically, the total deletion of ern0030 conferred a decrease in tetracycline MICs that was consistent with gene expression data showing no significant tet(M) induction under tetracycline SIC in ern0030-deleted mutant as opposed to a 10-fold increase of tet(M) expression in the wild-type strain. We investigated the transcriptional attenuation mechanism by toeprint assay. Whereas the expected tet(M) ribosome-binding site (RBS) was detected, the RBS of the putative leader peptide was not highlighted by toeprint assay, suggesting the transcriptional attenuation was unlikely. Here, we demonstrate that Ern0030 has a role in regulation of tet(M) expression and propose a novel model of tet(M) regulation alternative or complementary to transcriptional attenuation.

Integrated metagenomic and metatranscriptomic analysis reveals actively expressed antibiotic resistomes in the plastisphere

The plastisphere is viewed as a reservoir for the antibiotic resistome in water environments and may pose health concerns. However, the expression profiles of the resistome in the plastisphere are largely unknown. Here, we profiled the occurrence, abundance, and transcriptional level of antibiotic resistance genes (ARGs), plasmid associated ARGs, microbial composition and ARG bacterial hosts in the plastisphere and urban river water using 16S rRNA gene sequencing, metagenomic sequencing, and metatranscriptomic sequencing methods. A total of 173 ARGs conferring resistance to 24 major classes of antibiotics commonly prescribed to humans and animals were detected in the plastisphere. Of these, 75 genes were observed with transcriptional activity, indicating that the antibiotic resistome in the plastisphere was not only present, but also actively expressed. Human pathogens belonging to family Enterobacteriaceae were identified as bacterial hosts of ARGs in the plastisphere. The opportunistic and multidrug resistant human pathogen Enterobacter cloacae was found to actively express tetG and confer tetracycline resistance to the plastisphere. Furthermore, 39 genes were identified as “plasmid associated ARGs” in the plastisphere, displaying a higher proportion of transcript abundance compared with water. The above results suggest that the plastisphere is a hotspot for antibiotic resistome acquisition, expression, and dissemination.

Playgrounds in City of Pushchino with Different Types of Coating as Reservoir of Antibiotic-Resistant Strains of Pseudomonas spp.

In this study, we investigated antibiotic-resistant microorganisms isolated by the direct plating method from 6 playgrounds in the city of Pushchino, Moscow Region, with different types of coating: sand, soil with sand, grass and a modern playground coating made of pressed rubber crumb. According to the results of the study, sand is the cleanest type of coating, both in terms of the total count of cultivated microorganisms (8 × 105/g of substrate) and in terms of the content of resistant strains. The most contaminated both in terms of the total count of cultivated microorganisms (1.2-1.9 × 109/g of substrate) and in terms of the content of antibiotic-resistant strains was the coating of pressed rubber crumb. We isolated 65 antibiotic-resistant strains of fluorescent pseudomonads. Nine Pseudomonas strains were found to contain antibiotic resistance plasmids (one belongs to P-1 incompatibility group, seven to IncP-7 and one to unidentified incompatibility group). For the first time, we discovered a conjugative plasmid pD4A-46 conferring tetracycline resistance and belonging to the P-7 incompatibility group. Taking into account the results obtained under this study, it can be recommended to periodically treat the crumb rubber coating with non-toxic antiseptics, i.e. hydrogen peroxide or chlorhexidine.

The effect of Candida famata and Lactobacillus plantarum on the number of coliforms and the antibiotic resistance and virulence of Escherichia coli in the gut of broilers

This study was undertaken to determine the effect of a yeast (Candida famata) and a bacterium (Lactobacillus plantarum), administered alone or in combination in the drinking water, on the population of yeast, Lactobacillus sp. and coliforms, and the prevalence of antimicrobial resistance (AMR) and virulence genes in Escherichia coli (E. coli) isolated from digesta samples taken throughout the life of broiler chickens. Male (Ross 308) day-old chicks (220) were used. C. famata (isolated from a chicken) and L. plantarum (isolated from a pig) were administered via the drinking water. Water was provided either untreated or with C. famata (CF; 108/ml), L. plantarum (LP; 105–108/ml), or a combination of CF and LP (106–108/ml) in water hoppers on 2 days each week for 35 days. Administering probiotics did not affect the growth performance in broiler chickens. No significant interactions were observed between main effects, and neither CF nor LP had any effect on the population size of Lactobacillus sp. or coliforms. The administration of C. famata increased the population density of yeasts in the small intestine at these ages. The population density of coliforms, Lactobacillus sp. and yeast decreased with age (P < 0.001). There was no significant effect of probiotics on the prevalence of phenotypic AMR and virulence genes in these studies. The prevalence of E. coli that was resistant to ampicillin and tetracycline, as well as carrying ≥3 virulence-associated genes, was greatest at the end of the starter phase (around 8 days old), before declining through the grower and finisher phases. There was only limited evidence that administering either CF or LP affected either the AMR or the virulence of E. coli in the bird. However, tetracycline resistance in E. coli was associated (P < 0.001, P < 0.01, P < 0.05, and P < 0.05) with the carriage of the iron uptake systems of E. coli D, iron-repressible protein, increased serum survival and temperature-sensitive haemagglutinin genes respectively, suggesting that the accumulation of iron and the genetic element conferring tetracycline resistance may be intertwined.

Directed Recovery and Molecular Characterization of Antibiotic Resistance Plasmids from Cheese Bacteria.

Resistance to antimicrobials is a growing problem of worldwide concern. Plasmids are thought to be major drivers of antibiotic resistance spread. The present work reports a simple way to recover replicative plasmids conferring antibiotic resistance from the bacteria in cheese. Purified plasmid DNA from colonies grown in the presence of tetracycline and erythromycin was introduced into plasmid-free strains of Lactococcus lactis, Lactiplantibacillus plantarum and Lacticaseibacillus casei. Following antibiotic selection, the plasmids from resistant transformants were isolated, analyzed by restriction enzyme digestion, and sequenced. Seven patterns were obtained for the tetracycline-resistant colonies, five from L. lactis, and one each from the lactobacilli strains, as well as a single digestion profile for the erythromycin-resistant transformants obtained in L. lactis. Sequence analysis respectively identified tet(S) and ermB in the tetracycline- and erythromycin-resistance plasmids from L. lactis. No dedicated resistance genes were detected in plasmids conferring tetracycline resistance to L. casei and L. plantarum. The present results highlight the usefulness of the proposed methodology for isolating functional plasmids that confer antibiotic resistance to LAB species, widen our knowledge of antibiotic resistance in the bacteria that inhabit cheese, and emphasize the leading role of plasmids in the spread of resistance genes via the food chain.

A novel plasmid from Aerococcus urinaeequi of porcine origin co-harboring the tetracycline resistance genes tet(58) and tet(61)

Tetracyclines are the broad-spectrum agents used in veterinary medicine and food animal production. Known mechanisms of tetracycline resistance include ribosome protection, active efflux and enzymatic inactivation. However, the presence of two different tet genes conferring different resistance mechanisms on the same plasmid has rarely been reported. In this study, we identified the tandem tetracycline resistance genes tet(61)-tet(58) on the novel plasmid pT4303. These tet genes were identified for the first time in Aerococcus urinaeequi. Reduced susceptibility to doxycycline was observed in S. aureus RN4220 harboring tet(61) when an extra tet(58) was expressed. Plasmid pT4303 was electrotransformed into S. aureus RN4220, E. faecalis JH2−2, S. suis BAA and E. coli DH5α and conferred tetracycline resistance (MIC ≥ 16) in both Gram-positive and Gram-negative bacteria, assuming that it might serve as a vehicle for the dissemination of the tetracycline resistance genes tet(61) and tet(58).

Detection of Tn916 Conferring Tetracycline Resistance in Clinical Isolates of Streptococcus pyogenes

Background: During the past twenty years, tetracycline resistance average has increased in Streptococcuspyogenes in many countries. Pneumococcal resistance to erythromycin and tetracycline is associated withthe insertion of the erm(B) into the transposons of the Tn916 family. To get datum that may be beneficial inresolving the diffusion of antimicrobial resistance, therefore, we can be specified antibiotic resistant genesand their corporation with mobile genetic elements. This study was proceed to explore the genetic regulationof Tn916-carrying tet(M) in clinical isolates of S.pyogenes . Methods: A twenty two of S.pyogenes isolateswere assemble from patients suffering from upper respiratory infection, and the susceptibility of theseisolates to tetracycline antibiotics was examined. Molecular detection of Tn916 was carried out by employcertain primers to amplify tet(M) gene in each isolates.Results: The results appeared that the resistance of the tetracycline group was 68.1%, 54.5%, 36.3% and31.8% for minocycline, tetracycline, oxytetracycline and doxycycline, respectively. Genetic analysis showedthat Tn916 was detected in eight of the S.pyogenes clinical isolates resistant to tetracycline. Conclusions:Our findings suggest that clinical isolates of S. pyogenes harboring a copy(s) of Tn916 conferring tetracyclineresistance. One possible explanation for resistance to tetracycline in these isolates is due to tet gene, whichwas most likely located on Tn916.

Microarray based genetic profiling of Staphylococcus aureus isolated from abattoir byproducts of pork origin.

Many parts of pork meat processing are currently not used for human consumption in Switzerland, although they are of great nutritional value. Therefore, data on the occurrence of pathogenic organisms on byproducts is extremely scarce and the prevalence and population structure of Staphylococcus aureus on meat processing sidestreams is unknown. Hence, abattoir byproducts of pork origin including ear, forefoot, heart, intestine, liver, rib bone, sternum, bladder, stomach, hind foot and tongue originating from six abattoirs were screened for S. aureus. The obtained isolates were investigated by spa typing and DNA microarray analysis to reveal their genomic profile and population structure. The prevalence of S. aureus was generally low with a mean of 8%. In total, 40 S. aureus strains were detected and assigned to 12 spa types (t015, t1491, t1778, t091, t337, t899, t2922, t7439, t1333, t208, t4049, t034) and seven clonal complexes (CC1, CC7, CC9, CC30, CC45, CC49, CC398). Detected enterotoxin genes included sea, seb, sec, seh, sel and egc encoded toxin genes seg, sei, sem, sen, seo, and seu. None of the isolates harbored genes conferring methicillin resistance, but blaZ/I/R genes causing penicillin resistance were frequently found. In addition, strains from CC398 exhibited tetM and tetK, conferring tetracycline resistance. Similarity calculations based on microarray profiles revealed no association of clonal complexes with particular body parts, but revealed a certain correspondence of clonal complex and originating abattoir.

Transfer of a mobile Staphylococcus saprophyticus plasmid isolated from fermented seafood that confers tetracycline resistance.

The complete nucleotide sequence of a tetracycline-resistance gene (tetK)-carrying plasmid from a Staphylococcus saprophyticus isolate from jeotgal, a Korean high-salt-fermented seafood, was determined. The plasmid, designated pSSTET1, was 4439 bp in length and encoded typical elements found in plasmids that replicate via a rolling-circle mechanism, including the replication protein gene (rep), a double-stranded origin of replication, a single-stranded origin of replication, and a counter-transcribed RNA sequence. Additionally, the plasmid recombination enzyme gene (pre), which may be involved in inter-plasmid recombination and conjugation, was found. Each gene exhibited >94% sequence identity with those harbored in other Staphylococcus species. pSSTET1 was conditionally transferred to Staphylococcus species in a host-dependent manner and transferred to an Enterococcus faecalis strain in vitro. Antibiotic susceptibility of the transconjugants was host-dependent and transconjugants maintained a tetracycline-resistant phenotype in the absence of selective pressure over 100 generations.

Antimicrobial susceptibility of bifidobacteria from probiotic milk products and determination of the genetic basis of tetracycline resistance in Enterococcus species after in vitro conjugation with Bifidobacterium animalis subsp. lactis

The study aimed at investigating the antimicrobial resistance (AMR) profiles of bifidobacteria from fermented milk products sold in the UK that are claimed to have beneficial effects on humans, and assess the potential transferability of AMR to other bacteria.Isolates of different Bifidobacterium species were screened for their susceptibility to 24 antimicrobials and the presence of AMR genes [tet(M), tet(L), tet(S), tet(Q), tet(K), tet(O), tet(W), aph(3″)-I, ant(2″)-I, aph(3″)-III, strA, strB, aadA, aadE, erm(A), erm(B), and erm(C)]. The ability of Bifidobacterium animalis subsp. lactis isolates to transfer the tet(W) gene to Enterococcus faecalis JH2-2 and Enterococcus faecium BM4105 was investigated by conjugation. Potential E. faecalis transconjugants (PETs) were analysed by whole genome sequencing (WGS) for the presence of tet(W), other DNA fragments from the donor and single-nucleotide polymorphisms (SNPs). A high level of resistance to aminoglycosides and a moderate level of resistance to tetracycline were observed, while susceptibility to macrolides, vancomycin, chloramphenicol and beta-lactams was determined. Only the tet(W) gene was detected in all B. animalis subsp. lactis isolates. Some PETs exhibited a significant tetracycline MIC increase from 1 to up to 16 mg/L. WGS did not detect tet(W) in the PETs, but SNPs in the rpsJ gene encoding the ribosomal protein S10, which is part of the 30S ribosomal subunit and contains a proposed tetracycline binding site. Substitutions (Tyr-58→Asp, Tyr-58→Cys, Asp-60→Tyr, Tyr-58→Ser, Ser-61→Tyr, Ala-54→Glu, Asp-60→Tyr) and an insertion of Thr between Thr55 and His56 were detected. No E. faecium potential transconjugants were recovered.B. animalis subsp. lactis isolates did not transfer the tet(W) gene to the Enterococcus species. The majority of the PETs had mutations within or near the ribosomal protein S10 vertex loop suggesting that mutations in the rpsJ gene confer tetracycline resistance to E. faecalis JH2-2.

Various Profiles of tet Genes Addition to tet(X) in Riemerella anatipestifer Isolates From Ducks in China.

To investigate tetracycline resistance and resistant genotype in Riemerella anatipestifer, the tetracycline susceptibility of 212 R. anatipestifer isolates from China between 2011 and 2017 was tested. The results showed that 192 of 212 (90.6%) R. anatipestifer isolates exhibited resistance to tetracycline (the MICs ranged from 4 to 256 μg/ml). The results of PCR detection showed that, 170 of 212 (80.2%) R. anatipestifer isolates possessed the tet(X) gene. Other genes, including tet(A), tet(M), tet(Q), tet(O), tet(B), and tet(O/W/32/O), were found at frequencies of 20.8, 4.7, 1.4, 0.9, 0.9, and 0.5%, respectively. However, tet(C), tet(E), tet(G), tet(K), and tet(W) were not detected in any isolate. In these tet gene positive strains, 31 (14.6%), 2 (0.9%), 5 (2.4%), 1 (0.5%), 3 (1.4%) were detected containing tet(A)/tet(X), tet(M)/tet(O), tet(M)/tet(X), tet(O)/tet(X), and tet(Q)/tet(X) simultaneously, respectively. One isolates, R131, unexpectedly contained three tet genes, i.e., tet(M), tet(O), and tet(X). Sequence analysis of the tet gene ORFs cloned from R. anatipestifer isolates confirmed that tet(A), tet(B), tet(M), tet(O), tet(Q) and an unusual mosaic tet gene tet(O/W/32/O) were present in R. anatipestifer. The MIC results of R. anatipestifer ATCC 11845 transconjugants carrying tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes exhibited tetracycline resistance with MIC values ranging from 4 to 64 μg/ml. Additionally, the tet(X) gene could transfer into susceptible strain via natural transformation (transformation frequencies of ~10−6). In conclusion, the tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes were found and conferred tetracycline resistance in R. anatipestifer isolates. Moreover, the tet(X) is the main mechanism of tetracycline resistance in R. anatipestifer isolates. To our knowledge, this is the first report of tet(A), tet(B), tet(M), tet(O), tet(Q), and mosaic gene tet(O/W/32/O) in R. anatipestifer.

Detection of antimicrobial resistance genes of Helicobacter pylori strains to clarithromycin, metronidazole, amoxicillin and tetracycline among Egyptian patients

BackgroundAntibiotic resistance of Helicobacter pylori (H. pylori) treatment is on the rise, and is affecting the efficacy of current used therapeutic regimens. AimWe aimed to enhance the understanding of antimicrobial resistance rates of H. pylori strains recovered from patients at Theodor Bilharz Research Institute Hospital in Egypt, as a mandatory step before starting treatment. Subjects and methodsMutant genes conferring metronidazole, amoxicillin, clarithromycin, and tetracycline resistance were detected in 60 H. pylori strains recovered from patients who underwent upper endoscopic examination. Patients were considered to be infected with H. pylori when rapid urease test and detection of 16S rRNA in gastric biopsies recorded positive. Molecular detection of resistant genes to metronidazole (rdx gene) and amoxicillin (pbp1A gene) was carried out by conventional PCR followed by sequencing of PCR products. While detection of 23S rRNA gene conferring clarithromycin resistance and detection of 16S rRNA mutation gene conferring tetracycline resistance were carried out by real-time PCR. ResultsH. pylori resistance rates to metronidazole, and amoxicillin were 25% and 18.3% respectively. While for clarithromycin and tetracycline, point mutations in 23S rRNA types A2142G and A2143G and in 16S rRNA of H. pylori were assessed by real time PCR assay respectively. Resistance mutant genes were found to be 6.7% of clarithromycin and 1.7% of tetracycline. Combined resistance rates to metronidazole and amoxicillin was (11.6%) followed by metronidazole and clarithromycin (5%), while patterns of clarithromycin and amoxicillin (1.6%), metronidazole, clarithromycin and amoxicillin (1.6%) were revealed. ConclusionData concerning antimicrobial resistance genes play an important role in empiric treatment of H. pylori infection. According to our results, H. pylori resistance to metronidazole and amoxicillin was relatively high. Clarithromycin is still a good option for first line anti-H. pylori treatment. Combined resistant strains are emerging and may have an effect on the combination therapy.

Tet(C) Gene Transfer between Chlamydia suis Strains Occurs by Homologous Recombination after Co-infection: Implications for Spread of Tetracycline-Resistance among Chlamydiaceae.

Chlamydia suis is a swine pathogen that has also recently been found to cause zoonotic infections of the human eye, pharynx, and gastrointestinal tract. Many strains contain a tetracycline class C gene [tet(C)] cassette that confers tetracycline resistance. The cassette was likely originally acquired by horizontal gene transfer from a Gram-negative donor after the introduction of tetracycline into animal feed in the 1950s. Various research groups have described the capacity for different Chlamydia species to exchange DNA by homologous recombination. Since over 90% of C. suis strains are tetracycline resistant, they represent a potential source for antibiotic-resistance spread within and between Chlamydiaceae species. Here, we examined the genetics of tet(C)-transfer among C. suis strains. Tetracycline-sensitive C. suis strain S45 was simultaneously or sequentially co-infected with tetracycline-resistant C. suis strains in McCoy cells. Potential recombinants were clonally purified by a harvest assay derived from the classic plaque assay. C. suis strain Rogers132, lacking transposases IS200 and IS605, was the most efficient donor, producing two unique recombinants detected in three of the 56 (5.4%) clones screened. Recombinants were found to have a minimal inhibitory concentration (MIC) of 8-16 μg/mL for tetracycline. Resistance remained stable over 10 passages as long as recombinants were initially grown in tetracycline at twice the MIC of S45 (0.032 μg/mL). Genomic analysis revealed that tet(C) had integrated into the S45 genome by homologous recombination at two unique sites depending on the recombinant: a 55 kb exchange between nrqF and pckG, and a 175 kb exchange between kdsA and cysQ. Neither site was associated with inverted repeats or motifs associated with recombination hotspots. Our findings show that cassette transfer into S45 has low frequency, does not require IS200/IS605 transposases, is stable if initially grown in tetracycline, and results in multiple genomic configurations. We provide a model for stable cassette transfer to better understand the capability for cassette acquisition by Chlamydiaceae species that infect humans, a matter of public health importance.

Prevalence of Chlamydia trachomatis, Ureaplasma spp., Mycoplasma genitalium and Mycoplasma hominis among outpatients in central Greece: absence of tetracycline resistance gene tet(M) over a 4-year period study.

A total of 301 men and women attending local urologists and gynaecologists in the state of Thessaly, central Greece, were tested for Chlamydia trachomatis, Ureaplasma spp., Mycoplasma genitalium and Mycoplasma hominis DNA. Investigation of the tet(M) gene, which confers tetracycline resistance in these genera, was also performed. Low incidence of C. trachomatis and Mycoplasma spp. as well as high prevalence of Ureaplasma spp., especially among women, were found. The tet(M) gene was absent in all cases, notably in a region where doxycycline administration remains the first therapeutic option unless special medical conditions direct otherwise.