Tet Genes Research Articles

A nisin-inducible chromosomal gene expression system based on ICE Tn5253 of Streptococcus pneumoniae, transferable among streptococci and enterococci

The present work reports the development and validation of a chromosomal expression system in Streptococcus pneumoniae which permits gene expression under the control of Lactococcus lactis lantibiotic nisin. The system is based on the integrative and conjugative element (ICE) Tn5253 of S. pneumoniae capable of site-specific chromosomal integration and conjugal transfer to a variety of bacterial species. We constructed an insertion vector that integrates in Tn5251, an ICE contained in Tn5253, which carries the tetracycline resistance tet(M) gene. The vector contains the nisRK regulatory system operon, the L. lactis nisin inducible promoter PnisA upstream of a multiple cloning site for target DNA insertion, and is flanked by two DNA regions of Tn5251 which drive homologous recombination in ICE Tn5253. For system evaluation, the emm6.1::ha1 fusion gene was cloned and integrated into the chromosome of the Tn5253-carrying pneumococcal strain FR24 by transformation. This gene encodes a fusion protein containing the signal peptide, the 122 N-terminal and the 140 C-terminal aa of the Streptococcus pyogenes M6 surface protein joined to the HA1 subunit of the influenza virus A hemagglutinin. Quantitative RT-PCR analysis carried out on total RNA purified from nisin treated and untreated cultures showed an increase in emm6.1::ha1 transcript copy number with growing nisin concentration. The expression of M6-HA1 protein was detected by Western blot and quantified by Dot blot, while Flow cytometry analysis confirmed the presence on the pneumococcal surface. Recombinant ICE Tn5253::[nisRK]-[emm6.1::ha1] containing the nisin-inducible expression system was successfully transferred by conjugation in different streptococcal species including Streptococcus gordonii, S. pyogenes, Streptococcus agalactiae and Enterococcus faecalis. As for S. pneumoniae, the emm6.1::ha1 transcript copy number and the amount of M6-HA1 protein produced correlated with the nisin concentration used for induction in all investigated bacterial hosts. We demonstrated that this host-vector expression system is stably integrated as a single copy within the bacterial chromosome, is transferable to both transformable and non transformable bacterial species, and allows fine tuning of protein expression modulated by nisin concentration. These characteristics make our system suitable for a wide range of applications including complementation assays, physiological studies, host-pathogen interaction studies.

Emergence of plasmid-borne tet(X4) resistance gene in clinical isolate of eravacycline- and omadacycline-resistant Klebsiella pneumoniae ST485.

Omadacycline and eravacycline are gradually being used as new tetracycline antibiotics for the clinical treatment of Gram-negative pathogens. Affected by various tetracycline-inactivating enzymes, there have been reports of resistance to eravacycline and omadacycline in recent years. We isolated a strain carrying the mobile tigecycline resistance gene tet(X4) from the feces of a patient in Zhejiang Province, China. The strain belongs to the rare ST485 sequence type. The isolate was identified as Klebsiella pneumoniae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The MICs of antimicrobial agents were determined using either the agar dilution method or the micro broth dilution method. The result showed that the isolate was resistant to eravacycline (MIC = 32 mg/L), omadacycline (MIC > 64 mg/L), and tigecycline (MIC > 32 mg/L). Whole-genome sequencing revealed that the tet(X4) resistance gene is located on the IncFII(pCRY) conjugative plasmid. tet(X4) is flanked by ISVsa3, and we hypothesize that this association contributes to the spread of the resistance gene. Plasmids were analyzed by S1-nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blotting, and electrotransformation experiment. We successfully transferred the plasmid carrying tet(X4) to the recipient bacteria by electrotransformation experiment. Compared with the DH-5α, the MICs of the transformant L3995-DH5α were increased by eight-fold for eravacycline and two-fold higher for omadacycline. Overall, the emergence of plasmid-borne tet(X4) resistance gene in a clinical isolate of K. pneumoniae ST485 underscores the essential requirement for the ongoing monitoring of tet(X4) to prevent and control its further dissemination in China.IMPORTANCEThere are still limited reports on Klebsiella pneumoniae strains harboring tetracycline-resistant genes in China, and K. pneumoniae L3995hy adds a new example to those positive for the tet(X4) gene. Importantly, our study raises concerns that plasmid-mediated resistance to omadacycline and eravacycline may spread further to a variety of ecological and clinical pathogens, limiting the choice of medication for extensively drug-resistant bacterial infections. Therefore, it is important to continue to monitor the prevalence and spread of tet(X4) and other tetracyclines resistance genes in K. pneumoniae and diverse bacterial populations.

Detection of Antibiotic Resistance Genes in Escherichia coli Isolated from Chicken Eggs

Total 150 chicken eggs were randomly collected from farms, households, and markets of Rajshahi and Naogaon districts. Isolation and identification of E. coli was done by using cultural and staining characteristics, and biochemical properties. Antibiotic sensitivity assay was also performed for the isolated E. coli by using disc diffusion method. Characterization and detection of antibiotic resistance genes in E. coli isolates were done through PCR analysis. Prevalence of E. coli was 30.00% in chicken eggs in Rajshahi and Naogaon districts. Results of antibiotic sensitivity patterns of the isolated E. coli showed 57.78%, 33.33%, 31.11%, 28.89%, 26.67%, 17.78%, 15.56%, 13.33%, 13.33%, and 11.11% resistance to ciprofloxacin, doxycycline, oxytetracycline, ceftriaxone, amoxycillin, erythromycin, levofloxacin, enrofloxacin, gentamycin, and neomycin, respectively. The isolates also showed 94.44%, 82.22%, 80.00%, 77.78%, 66.67%, 60.00%, 55.56%, 48.89%, 46.67%, and 33.33% sensitive to neomycin, enrofloxacin, erythromycin, levofloxacin, gentamycin, ceftriaxone, oxytetracycline, doxycycline, amoxicillin, and ciprofloxacin, respectively. All 45 phenotypic identified isolates were confirmed as E. coli by PCR analysis. Genotypic identified isolates of E. coli were further characterized by PCR to detect antibiotic resistance genes. Out of 45 E. coli isolates 37 (82.22%) showed tetracycline resistance (tetA) gene, 32 (71.11%) showed tetracycline resistance (tetB) gene, and interestingly all 45 (100%) isolates showed quinolone group resistance (gyrA) gene by PCR analysis. J. of Sci. and Tech. Res. 5(1): 135-142, 2023

Synonymous codon substitutions modulate transcription and translation of a divergent upstream gene by modulating antisense RNA production

Synonymous codons were originally viewed as interchangeable, with no phenotypic consequences. However, substantial evidence has now demonstrated that synonymous substitutions can perturb a variety of gene expression and protein homeostasis mechanisms, including translational efficiency, translational fidelity, and cotranslational folding of the encoded protein. To date, most studies of synonymous codon-derived perturbations have focused on effects within a single gene. Here, we show that synonymous codon substitutions made far within the coding sequence of Escherichia coli plasmid-encoded chloramphenicol acetyltransferase (cat) can significantly increase expression of the divergent upstream tetracycline resistance gene, tetR. In four out of nine synonymously recoded cat sequences tested, expression of the upstream tetR gene was significantly elevated due to transcription of a long antisense RNA (asRNA) originating from a transcription start site within cat. Surprisingly, transcription of this asRNA readily bypassed the native tet transcriptional repression mechanism. Even more surprisingly, accumulation of the TetR protein correlated with the level of asRNA, rather than total tetR RNA. These effects of synonymous codon substitutions on transcription and translation of a neighboring gene suggest that synonymous codon usage in bacteria may be under selection to both preserve the amino acid sequence of the encoded gene and avoid DNA sequence elements that can significantly perturb expression of neighboring genes. Avoiding such sequences may be especially important in plasmids and prokaryotic genomes, where genes and regulatory elements are often densely packed. Similar considerations may apply to the design of genetic circuits for synthetic biology applications.

Emergence of Staphylococcus argenteus in pediatrics: Molecular insights from a hospital in East China.

Staphylococcus argenteus is a novel species within the Staphylococcus aureus complex and can cause serious bloodstream infections (BSIs) in humans, which have been mainly reported in adults, especially the elderly. In this study, we analyzed the molecular characterization of a strain of S. argenteus (22WJ8192) isolated from the peripheral vein blood sample of a seven-month-old female infant in Eastern China. The 22WJ8192 belonged to sequence type (ST)2250 and harbored six antibiotic-resistance genes and 53 virulence genes and was resistant to penicillin. Additionally, we conducted a comparative analysis of the molecular characteristics of S. argenteus sourced from various origins within the dataset, predominantly from the National Center for Biotechnology Information Collection (NCBI) genome database. Antibiotic-resistance genes blaR1, blaI_of_Z, blaZ, fosB-Saur, tet(L), aph(3")-IIIa, mecA, and dfrG were more prevalent among the strains of human origin. Virulence genes lukF-PV, sak, sdrE, scn, sdrC, and sdrD were more prevalent among strains of human origin. The presence of antibiotic-resistance genes blaR1, blaI_of_Z, blaZ, fosB-Saur, and aph(3")-IIIa in strain 22WJ8192 was also more common among strains of human origin in the dataset. Conversely, the antibiotic-resistance genes tet(L), mecA, and dfrG, typically found in strains of human origin, were not detected in 22WJ8192. Additionally, virulence genes lukF-PV, sak, sdrE, scn, sdrC, and sdrD present in 22WJ8192 exhibited a higher prevalence among strains of human origin in the dataset. In conclusion, this study emphasizes the potential of S. argenteus ST2250 to induce severe bloodstream infections in infants, shedding light on the molecular characteristics of this strain.

Prevalence and Genomic Characterization of Multidrug-Resistant Salmonella enterica Serovar Kentucky Sequence Type 198 Circulating - Beijing Municipality, China, 2016-2023.

Highly fluoroquinolone-resistant Salmonella enterica serovar Kentucky (S. Kentucky) of sequence type (ST) 198 has emerged as a global multidrug-resistant (MDR) clone, posing a threat to public health. Whole genome sequencing and antibiotic susceptibility testing was used to characterize the population structure and evolutionary history of 54 S. Kentucky isolates recovered from food and human clinical cases in Beijing from 2016 to 2023. All 54 S. Kentucky ST198 isolates exhibited resistance to quinolones, carrying point mutations in the quinolone resistance-determining regions (gyrA_S83F and parC_S80I). Resistance to other antibiotics (folate pathway inhibitors, cephems, aminoglycosides, phenicols, rifamycin, fosfomycin, macrolides, and tetracyclines), mediated by the sul1, sul2, dfrA14, bla CTX-M, bla TEM-1B, aac(3)-Id, aadA2, aadA7, aph(3')-I, aph(3'')-Ib, rmtB, floR, arr-2, fosA, mph(A), and tet(A) genes, was also observed in different combinations. The Beijing S. Kentucky ST198 evolutionary tree was divided into clades 198.2-1 and 198.2-2, which were further differentiated into three subclades: 198.2-2A, 198.2-2B, and 198.2-2C. Compared with the extended-spectrum β-lactamase-encoding gene bla CTX-M-14b in 198.2-1, the co-existence of bla CTX-M-55 and bla TEM-1B, as well as chromosomally located qnrS1, was detected in most 198.2-2 isolates, which showed more complex MDR phenotypes. S. Kentucky ST198 outbreak isolates derived from two predominant clonal sources: 198.2-1 with cgST236434 and 198.2-2A with cgST296405. The S. Kentucky population in Beijing is genetically diverse, consisting of multiple co-circulating lineages that have persisted since 2016. Strengthening surveillance of food and humans will aid in implementing measures to prevent and control the spread of AMR.

Genomic characterization of methicillin-resistant Staphylococcus aureus isolated from patients attending regional referral hospitals in Tanzania

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) colonization increases the risk of subsequent infection by MRSA strain complex interlinking between hospital and community-acquired MRSA which increases the chance of drug resistance and severity of the disease.ObjectiveGenomic characterization of Staphylococcus aures strains isolated from patients attending regional referral hospitals in Tanzania.MethodologyA laboratory-based cross-sectional study using short read-based sequencing technology, (Nextseq550,Illumina, Inc. San diego, California, USA). The samples used were collected from patients attending selected regional referral hospitals in Tanzania under the SeqAfrica project. Sequences were analyzed using tools available in the center for genomic and epidemiology server, and visualization of the phylogenetic tree was performed in ITOL 6.0. SPSS 28.0 was used for statistical analysis.ResultsAmong 103 sequences of S. aureus, 48.5% (50/103) carry the mecA gene for MRSA. High proportions of MRSA were observed among participants aged between 18 and 34 years (52.4%), in females (54.3%), and among outpatients (60.5%). The majority of observed MRSA carried plasmids rep5a (92.0%), rep16 (90.0%), rep7c (90.0%), rep15 (82.0%), rep19 (80.0%) and rep10 (72.0%). Among all plasmids observed rep5a, rep16, rep20, and repUS70 carried the blaZ gene, rep10 carried the erm(C) gene and rep7a carried the tet(K) gene. MLST and phylogeny analysis reveal high diversity among MRSA. Six different clones were observed circulating at selected regional hospitals and MRSA with ST8 was dominant.ConclusionThe study reveals a significant presence of MRSA in Staphylococcus aureus strains from Tanzanian regional hospitals, with nearly half carrying the mecA gene. MRSA is notably prevalent among young adults, females, and outpatients, showing high genetic diversity and dominance of ST8. Various plasmids carrying resistance genes indicate a complex resistance profile, highlighting the need for targeted interventions to manage MRSA infections in Tanzania.

Hyperthermophilic composting of livestock waste drastically reduces antimicrobial resistance

Composting is the most common method for managing livestock waste. However, it often fails to effectively eliminate antimicrobial resistance, resulting in potential antimicrobial resistance in livestock waste. Hence, a more effective livestock waste management strategy is warranted. The hyperthermophilic composting method uses recycled compost containing hyperthermophilic microbes to raise the compost temperature up to approximately 100 °C (compost temperature in normal composting: 50–70 °C). This study clarifies the effectiveness of hyperthermophilic composting method in attenuating antimicrobial resistance during livestock waste composting in both a composting simulator and on a field dairy farm. We analyzed bacterial abundance and community composition, performed PCR analysis, and evaluated the concentration of residual antimicrobials in the compost. Hyperthermophilic composting significantly reduced the abundance of culturable bacteria and Escherichia coli including that of antimicrobial-resistant culturable bacteria and E. coli in both the simulated and field dairy farms. The copy numbers of the tested antimicrobial resistance genes (tetA, tetB, blaTEM, and blaCTX-M) decreased substantially. Residual antimicrobials (tetracyclines and β-lactams) were not detected in the field dairy farms, and network analysis showed that potential hosts of antimicrobial-resistance genes were eliminated. These results indicate that hyperthermophilic composting significantly reduces the abundance of antimicrobial-resistant bacteria, antimicrobial-resistance genes, and residual antimicrobials at low cost and easy operation while generating valuable agricultural resource as the final output.

Molecular Basis of Antimicrobial Resistance in Group B Streptococcus Clinical Isolates from Saudi Arabia.

Published data on the molecular mechanisms underlying antimicrobial resistance in Group B Streptococcus (GBS) isolates from Saudi Arabia are lacking. Here, we aimed to determine the genetic basis of resistance to relevant antibiotics in a collection of GBS clinical isolates (n = 204) recovered from colonized adults or infected patients and expressing serotypes Ia, Ib, II, III, V, and VI. Initial susceptibility testing revealed resistance to tetracycline (76.47%, n = 156/204), erythromycin (36.76%, n = 75/204), clindamycin (25.49%, n = 52/204), levofloxacin (6.37%, n = 13/204), and gentamicin (2.45%, n = 5/204). Primers designed for the detection of known resistance determinants in GBS identified the presence of erm(A), erm(B), mef(A), and/or lsa(C) genes at the origin of resistance to macrolides and/or clindamycin. Of these, erm(B) and erm(A) were associated with the cMLSB (n = 46) and iMLSB (n = 28) phenotypes, respectively, while mef(A) was linked to the M phenotype (n = 1) and lsa(C) was present in isolates with the L phenotype (n = 8). Resistance to tetracycline was mainly mediated by tet(M) alone (n = 112) or in combination with tet(O) (n = 10); the remaining isolates carried tet(O) (n = 29), tet(L) (n = 2), or both (n = 3). Isolates resistant to gentamicin (n = 5) carried aac(6')-Ie-aph(2')-Ia, and those exhibiting resistance to levofloxacin (n = 13) had alterations in GyrA and/or ParC. Most isolates with the erm gene (93.24%, n = 69/74) also had the tet gene and were therefore resistant to erythromycin, clindamycin, and tetracycline. Overall, there were no clear associations between serotypes and resistance genotypes except for the presence of erm(B) in serotype Ib isolates. Dissemination of antibiotic resistance genes across different serotypes represents a public health concern that requires further surveillance and appropriate antibiotic use in clinical practice.

Antibiotic Resistance Profiles of Escherichia coli and Salmonella spp. Isolated From Dairy Farms and Surroundings in a Rural Area of Western Anatolia, Turkey.

Background Antibiotic resistance is a significant public health issue worldwide. Antibiotic-resistant zoonotic bacteria such as Escherichia coli (E. coli), Campylobacter, Salmonella, Listeria, Coxiella, and Mycobacterium can be particularly isolated from biofertilizers. Epidemiological studies have shown that cases of foodborne infections and intoxications are significantly related to animal-derived foods. The presence of these species in aquatic environments indicates areas or organisms contaminated with animal or human feces. Especially, the presence of E. coli in aquatic environments has become a serious problem worldwide. Pathogenic strains of E. coli cause waterborne and foodborne diseases. Materials and methods This study included a total of 290 samples collected from five different dairy farms between April and September 2023 which comprised 20 samples of cow manure, 20 samples of milk, three samples of dairy workers' hand washing water, five samples ofsoil, five samples of water, and five samples of vegetables. The samples taken from the farms were homogenized with 0.1% peptone water at a ratio of 1/10. They were then cultured on xylose lysine deoxycholate (XLD), eosin methylene blue agar (EMB), and blood agar media, and gram-negative colonies were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the VITEK2 automated system (BioMerieux Inc., Durham, NC). Amplification of the isolated DNA extracts was performed with A.B.T.™ 2X HS-PCR MasterMix (A.B.T Laboratory Industry, Arnavutköy, Turkey) in the SimpliAmp™ thermal cycler (Thermo Fischer Scientific Inc., Waltham, MA) and visualized by agarose gel electrophoresis. Results Among the 52E. coli strains isolated in our study, the highest antibiotic sensitivity rate was observed in meropenem, while the lowest sensitivity rates were determined in cefazolin and cefuroxime. While two of the Salmonella spp. (n = 2) isolates were found to be resistant to tetracycline, and one was found to be resistant to penicillin and ampicillin. No resistance to trimethoprim/sulfamethoxazole was detected in either isolate. Extended-spectrum beta-lactamases (ESBLs) were detected in only four (7.7%) E. coli strains. While tetA, tetB, and TEM genes were seen in almost all E. coli strains, they were not found in Salmonella spp. Conclusion In conclusion, our study revealed the presence of antimicrobial resistance genes in E. coli and Salmonella spp. isolates collected from various farms and environmental samples, which render the antimicrobials used for disease treatment ineffective. Consequently, research should be undertaken to prevent the development of new resistance genes in our country, as creating new medications and treatment strategies for these diseases is costly and time-intensive.

Characterization of a ST137 multidrug-resistant Campylobacter jejuni strain with a tet(O)-positive genomic island from a bloodstream infection patient

Campylobacter jejuni (C. jejuni) is a major cause of gastroenteritis and rarely cause bloodstream infection. Herein, we characterized a multidrug-resistant C. jejuni strain LZCJ isolated from a tumor patient with bloodstream infection. LZCJ was resistant to norfloxacin, ampicillin, ceftriaxone, ciprofloxacin and tetracycline. It showed high survival rate in serum and acidic environment. Whole genome sequencing (WGS) analysis revealed that strain LZCJ had a single chromosome of 1,629,078 bp (30.6 % G + C content) and belonged to the ST137 lineage. LZCJ shared the highest identity of 99.66 % with the chicken-derived C. jejuni MTVDSCj20. Four antimicrobial resistance genes (ARGs) were detected, blaOXA-61, tet(O), gyrA (T86I), and cmeR (G144D and S207G). In addition, a 12,746 bp genomic island GI_LZCJ carrying 15 open reading frames (ORFs) including the resistance gene tet(O) was identified. Sequence analysis found that the GI_LZCJ was highly similar to the duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. 137 non-synonymous mutations in motility related genes (flgF, fapR, flgS), capsular polysaccharide (CPS) coding genes (kpsE, kpsF, kpsM, kpsT), metabolism associated genes (nuoF, nuoG, epsJ, holB), and transporter related genes (comEA, gene0911) were confirmed in LZCJ compared with the best closed chicken-derived strain MTVDSCj20. Our study showed that C. jejuni strain LZCJ was highly similar to the chicken-derived strain MTVDSCj20 but with a lot of SNPs involved in motility, CPS and metabolism coding genes. This strain possessed a tet(O)-positive genomic island GI_LZCJ, which was closed to duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. The above data indicated that the LZCJ strain may originate from foodborne bacteria on animals and the importance of continuous surveillance for the spread of foodborne bacteria.

Identification of novel Tet(X6)-Tet(X2) recombinant variant in Elizabethkingia meningoseptica from a bullfrog farm and downstream river in China.

The dissemination of strains producing tetracyclines monooxygenase Tet(X) from breeding farms to the natural environment poses a potential threat to public health. Antimicrobial susceptibility testing and WGS were performed to identify resistance phenotypes and genotypes. Cloning experiments, sequence alignment, and homology modeling were used to characterize the function and formation mechanisms of the recombinant variant. The mobilization potential of Tet(X) was assessed by collinearity analysis, conjugation experiments, and phylogenetic analysis. Three tet(X)-producing Elizabethkingia meningoseptica strains were isolated from bullfrog breeding ponds, the sewage outlet, and downstream river in Zhejiang Province, China. These strains carry a novel Tet(X) variant, differing from Tet(X6) by seven residues, and possess the ability to degrade tetracyclines. Interestingly, the novel Tet(X) is a recombinant variant formed by homologous recombination of Tet(X6) and the C-terminal of Tet(X2). Further analysis revealed that Tet(X6) formed several Tet(X) variants, including Tet(X5), through homologous recombination. The novel tet(X) gene is located on a circularizable integrative and conjugative element (ICEEmeChn3), with ISwz1 participating in the recombination of its multi-drug resistance region, potentially facilitating the mobilization and recombination of tet(X) in early hosts. These three strains were clonally transmitted and shared a close genetic relationship (SNP < 62) with a clinically-sourced strain isolated from the same province. To our knowledge, this is the first report of homologous recombination between Tet(X) variants with differing activities. These clonal strains provide evidence of the transmission of tet(X)-positive strains from aquaculture sewage to the natural environment, highlighting the need to strengthen the monitoring and management of this emerging farming model.

Differential drivers on the occurrence of antibiotic resistance genes in agricultural soils and crops: Evidence from the different fertilization regimes

Organic fertilizer application caused bacterial resistance contamination in farming systems has been widely documented, and long-term fertilization will exacerbate the migration of antibiotic resistance genes (ARGs) to crops and humans. However, it remains unclear whether a combined pattern of chemical and organic fertilizer application can arrest the high prevalence of ARGs in soil-crop. Here we investigated the ARGs occurrence under different fertilization regimes, and explored the mechanisms by which micro-ecological shifts and geochemical factors in modulating the ARGs fate in soil and crop. The results showed that the soil ARGs abundance was highest under 100% organic fertilizer, while the highest ARGs abundance in crops was observed at 100% chemical fertilizer. Application of organic fertilizers with more than 50% ration intensified the soil accumulation and migration of tetA, sul1, sul2 and macA genes. And, multidrug_transporter, macA and sul1 were co-shared in soil and crop, where ARGs potential hosts differed complete in the two, suggesting that these ARGs may be transferred across media by horizontal transfer. Procrustes analysis revealed that soil microbial community was significantly correlated with ARG hosts, and soil microbial evolutionary pathway was congruent with antibiotic resistance, suggesting that fertilizers affect soil ARGs abundance mainly by altering soil microbial composition and their ecological evolutionary trajectories. While, no significant correlation was observed between endophytes and crop ARG host. Structural equations demonstrated that soil nutrients and crop growth characteristics contributed largely to the prevalence of crop ARGs. This outcome will provide new insights into the high prevalence of ARGs in soil and crop, and offer fertilizer recommendations for effectively curbing antibiotic-resistance contamination in farming systems.

The swine waste resistome: Spreading and transfer of antibiotic resistance genes in Escherichia coli strains and the associated microbial communities

The overuse of antimicrobials in livestock farming has led to the development of resistant bacteria and the spread of antibiotic-resistant genes (ARGs) among animals. When manure containing these antibiotics is applied to agricultural fields, it creates a selective pressure that promotes the acquisition of ARGs by bacteria, primarily through horizontal gene transfer. Most research on ARGs focuses on their role in clinical antibiotic resistance and their transfer from environmental sources to bacteria associated with humans, such as Escherichia coli. The study investigates the spread of antibiotic-resistant genes (ARGs) through class 1 integrons in 27 Escherichia coli strains from pig manure. It focuses on six common ARGs (ermB, cmlA, floR, qnrS, tetA, and TEM) and the class 1 integron gene, assessing their prevalence in manure samples from three pig farms. The study found correlations and anticorrelations among these genes, indicating a predisposition of the integron in spreading certain ARGs. Specifically, cmlA and tetA genes were positively correlated with each other and negatively with int1, suggesting they are not transferred via Int1. Farm B had the highest int1 counts and a higher abundance of the TEM gene, but lower levels of cmlA and tetA genes. The results underscore the complexity of predicting ARG spread in agricultural environments and the associated health risks to humans through the food chain. The study's results offer valuable insights into the antibiotic-resistant genes (ARGs) profile in swine livestock, potentially aiding in the development of methods to trace ARGs in the environment.

Products from Photolysis Reactions of Tetracycline Mediated by Clay and Humic Substance Induce Contrasting Expressions of Target Resistance Genes.

Phototransformation is a key process affecting the fate of many antibiotics in the environment, but little is known about whether their photoproducts exert selective pressure on bacteria by inducing antibiotic resistance genes (ARGs). Here, we examined the expression of tetracycline resistance gene tet(M) of a fluorescent Escherichia coli whole-cell bioreporter influenced by the phototransformation of tetracycline. The presence of suspended smectite clay (montmorillonite or hectorite, 1.75 g/L) or dissolved humic substance (Pahokee Peat humic acid or Pahokee peat fulvic acid, 10 mg C/L) in aqueous solutions markedly facilitated the transformation of tetracycline (initially at 400 μg/L) with half-life shortened by 1.4-2.6 times. Despite the similar phototransformation ratios (80-90%) of the total loaded tetracycline after 60 min irradiation, the decreased ratios of cell fluorescence intensity (which was proportional to the expression amount of ARG tet(M)) were much higher with the two clays (94 and 93%) than with the two humic substances (44 and 69%) when compared to the respective dark controls. As illustrated by mass spectroscopic and chemical analyses, tetracycline was proposed to be mainly transformed to amide (ineffective in inducing ARGs) with the presence of clays by reaction with self-photosensitized singlet oxygen (1O2), while the humic substances might catalyze the production of another two demethylated and/or deaminated compounds (still effective in inducing ARGs) in addition to the amide compound via reaction with triplet excited state dissolved organic matter (3DOM*). As clay minerals and humic substances are important soil constituents and ubiquitously present in surface environments, the observed clay and humic-dependent photooxidation pathways of tetracycline and the differing selective pressures of the associated products highlight the need for monitoring the transformation compounds of antibiotics and provide critical insight into the development of antibiotic treatment protocols.

Antimicrobial susceptibilities, resistance mechanisms and molecular characteristics of toxigenic Clostridioides difficile isolates in a large teaching hospital in Chongqing, China

ObjectivesClostridioides difficile ranks among the primary sources of healthcare-related infections and diarrhoea in numerous nations. We evaluated the drug susceptibility and resistance mechanisms of C. difficile isolates from a hospital in Chongqing, China, and identified resistance rates and resistance mechanisms that differed from previous findings. MethodsThe toxin genes and drug resistance genes of clinical strains were detected using Polymerase Chain Reaction (PCR), and these strains were subjected to Multilocus Sequence Typing (MLST). The agar dilution technique was employed for assessing susceptibility of antibiotics. Clinical data collection was completed through a review of electronic medical records. ResultsA total of 67 strains of toxin-producing C. difficile were detected. All C. difficile isolates demonstrated susceptibility to both metronidazole and vancomycin. However, resistance was observed in 8.95%, 16.42%, 56.72%, 56.72%, 31.34% and 5.97% of the isolates for tigecycline, tetracycline, clindamycin, erythromycin, moxifloxacin and rifampin, respectively. Among the strains with toxin genotypes A + B + CDT - and belonging to the ST3, six strains exhibited reduced susceptibility to tigecycline (MIC=0.5mg/L) and tetracycline (MIC=8mg/L). The tetA(P) and tetB(P) genes were present in these six strains, but were absent in tetracycline-resistant strains. Resistance genes (ermB, tetM, tetA(P) and tetB(P)) and mutations (in gyrA, gyrB, and rpoB) were identified in resistant strains. ConclusionsIn contrast to prior studies, we found higher proportions of ST3 isolates with decreased tigecycline sensitivity, sharing similar resistance patterns and resistance genes. In the resistance process of tigecycline and tetracycline, the tetA(P) and tetB(P) genes may play a weak role.

Prevalence and characterization of an integrative and conjugative element carrying tet(X) gene in Elizabethkingia meningoseptica

ObjectivesTo investigate the tet(X) gene, a determinant of tigecycline resistance, in the emerging pathogen Elizabethkingia meningoseptica and its association with an integrative and conjugative element (ICE). MethodsAll E. meningoseptica genomes from the National Center for Biotechnology Information (n = 87) were retrieved and annotated for resistome searches using the CARD database. A phylogenic analysis was performed based on the E. meningoseptica core genome. The ICE was identified through comparative genomics with other ICEs occurring in Elizabethkingia spp. ResultsPhylogenetic analysis revealed E. meningoseptica genomes from six countries distributed across different lineages, some of which persisted for years. The common resistome of these genomes included blaBlaB, blaCME, blaGOB, ranA/B, aadS, and catB (genes associated with resistance to β-lactams, aminoglycosides, and chloramphenicol). Some genomes also presented additional resistance genes (dfrA, ereD, blaVEB, aadS, and tet(X)). Interestingly, tet(X) and aadS were located in an ICE of 49 769 bp (ICEEmSQ101), which was fully obtained from the E. meningoseptica SQ101 genome. We also showed evidence that the other 27 genomes harboured this ICE. The distribution of ICEEmSQ101, carrying tet(X), was restricted to a single Chinese lineage. ConclusionsThe tet(X) gene is not prevalent in the species E. meningoseptica, as previously stated for the genus Elizabethkingia, since it is present only in a single Chinese lineage. We identified that several E. meningoseptica genomes harboured an ICE that mobilized the Elizabethkingia tet(X) gene and exhibited characteristics similar to the ICEs of other Flavobacteria, which would favour their transmission in this bacterial family.

Unveiling the overlooked threat: antibiotic resistance in groundwater near an abandoned sulfuric acid plant in Xingyang, China.

Groundwater near a sulfuric acid plant in Xingyang, Henan, China was sampled from seven distinct sites to explore the prevalence of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). Results showed that genes aadA, blaCTX-M, tetA, qnrA, and sul1 were detected with 100% frequency followed by aac(6')-Ib (85.71%), ermB (85.71%), and tetX (71.42%). Most abundant ARGs were sul1 in LSA2 (1.15 × 1011 copies/mL), tetA in LSA6 (4.95 × 1010 copies/mL), aadA in LSA2 (4.56 × 109 copies/mL), blaCTX-M in LSA4 (1.19 × 109 copies/mL), and ermB in LSA5 (1.07 × 109 copies/mL). Moreover, in LSA2, intl1 as a marker of class 1 integron emerged as the most abundant gene as part of MGE (2.25 × 1011 copies/mL), trailed by ISCR1 (1.57 × 109 copies/mL). Environmental factors explained 81.34% of ARG variations, with a strong positive correlation between the intl2 and blaCTX-M genes, as well as the ISCR1 gene and qnrA, tetA, intl2, and blaCTX-M. Furthermore, the intI1 gene had a strong positive connection with the aadA, tetA, and sul1 genes. Moreover, the aac(6')-Ib gene was associated with As, Pb, Mg, Ca, and HCO3-. The intl2 gene was also shown to be strongly associated with Cd. Notably, network analysis highlighted blaCTX-M as the most frequently appearing gene across networks of at least five genera. Particularly, Lactobacillus, Plesiomonas, and Ligilactobacillus demonstrated correlations with aadA, qnrA, blaCTX-M, intI2, and ISCR1. Based on 16S rRNA sequencing, the dominant phyla were Proteobacteria, Firmicutes, Bacteroidota, Acidobacteriota, and Actinobacteriota, with dominant genera including Pseudomonas, Ligilactobacillus, Azoarcus, Vogesella, Streptococcus, Plesiomonas, and Ferritrophicum. These findings enhance our understanding of ARG distribution in groundwater, signaling substantial contamination by ARGs and potential risks to public health.

A promising metabolite, 9-aminominocycline, restores the sensitivity of tigecycline against tet(X4)-positive Escherichia coli.

The emergence and widespread of tigecycline resistance undoubtedly poses a serious threat to public health globally. The exploration of combination therapies has become preferred antibacterial strategies to alleviate this global burden. In this study, tigecycline-resistant tet(X4)-positive Escherichia coli were selected for adjuvant screening. Interestingly, 9-aminominocycline (9-AMC), one of the tigecycline metabolites, exhibits synergistic antibacterial activity with tigecycline using checkerboard assay. The efficacy in vitro and in vivo was evaluated, and the synergistic mechanism was further explored. The results suggested that 9-AMC combined with tigecycline could inhibit the growth of antibiotic resistant bacteria, efficiently retard the evolution of tet(X4) gene and narrow the drug mutant selection window. In addition, the combination of tigecycline and 9-AMC could destroy the normal membrane structure of bacteria, inhibit the formation of biofilm, remarkably reduce the level of intracellular ATP level, and accelerate the oxidative damage of bacteria. Furthermore, 9-AMC is more stable in the bind of Tet(X4) inactivating enzyme. The transcriptomics analysis revealed that the genes related to the 9-AMC and tigecycline were mainly enriched in ABC transporters. Collectively, the results reveal the potentiation effects on tigecycline and the probability of 9-AMC as a novel tigecycline adjuvant against tet(X4)-positive Escherichia coli, which provides new insights for adjuvant screening.

Fertilizing drug resistance: Dissemination of antibiotic resistance genes in soil and plant bacteria under bovine and swine slurry fertilization

The increasing global demand for food production emphasizes the use of organic animal fertilizers, such as manure and slurry, to support sustainable agricultural practices. However, recent studies highlight concerns about antibiotic resistance determinants in animal excrements, posing a potential risk of spreading antibiotic resistance genes (ARGs) in agricultural soil and, consequently, in food products.This study examines the dissemination of ARGs within the soil and plant-associated microbiomes in cherry radish following the application of swine and bovine slurry. In a 45-day pot experiment, slurry-amended soil, rhizospheric bacteria, and endophytic bacteria in radish roots and leaves were sampled and analyzed for 21 ARGs belonging to 7 Antibiotic Resistance Phenotypes (ARPs). The study also assessed slurry's impact on soil microbiome functional diversity, enzymatic activity, physicochemical soil parameters, and the concentration of 22 selected antimicrobials in soil and plant tissues.Tetracyclines and β-lactams were the most frequently identified ARGs in bovine and swine slurry, aligning with similar studies worldwide. Swine slurry showed a higher prevalence of ARGs in soil and plant-associated bacteria, particularly TET genes, reflecting pig antibiotic treatments. The persistent dominance of TET genes across slurry, soil, and plant microbiomes highlights significant influence of slurry application on gene occurrence in plant bacteria. The presence of ARGs in edible plant parts underscores health risks associated with raw vegetable consumption. Time-dependent dynamics of ARG occurrence highlighted their persistent presence throughout the experiment duration, influenced by the environmental factors and antibiotic residuals. Notably, ciprofloxacin, which was the only one antimicrobial detected in fertilized soil, significantly impacted bovine-amended variants. Soil salinity modifications induced by slurry application correlated with changes in ARG occurrence. Overall, the research underscores the complex relationships between agricultural practices, microbial activity, and antibiotic resistance dissemination, emphasizing the need for a more sustainable and health-conscious farming approaches.