Gramine sensitizes Klebsiella pneumoniae to tigecycline killing.

BACKGROUND: The presence of plasmid-mediated resistance-nodulation-division (RND) efflux pump gene cluster tmexCD1-toprJ1 and its related variants has been associated with heightened resistance to tigecycline, thus diminishing its effectiveness. In this study, we explored the potential of gramine, a naturally occurring indole alkaloid, as an innovative adjuvant to enhance the treatment of infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters. METHODS: The synergistic potential of gramine in combination with antibiotics against both planktonic and drug-tolerant multidrug-resistant Enterobacterales was evaluated using the checkerboard microbroth dilution technique and time-killing curve analyses. Afterwards, the proton motive force (PMF) of cell membrane, the function of efflux pump and the activity of antioxidant system were determined by fluorescence assay and RT-PCR. The intracellular accumulation of tigecycline was evaluated by HPLC-MS/MS. The respiration rate, bacterial ATP level and the NAD+/NADH ratio were investigated to reveal the metabolism state. Finally, the safety of gramine was assessed through hemolytic activity and cytotoxicity assays. Two animal infection models were used to evaluate the in vivo synergistic effect. RESULTS: Gramine significantly potentiated tigecycline and ciprofloxacin activity against tmexCD1-toprJ1 and its variants-positive pathogens. Importantly, the synergistic activity was also observed against bacteria in special physiological states such as biofilms and persister cells. The mechanism study showed that gramine possesses the capability to augment tigecycline accumulation within cells by disrupting the proton motive force (PMF) and inhibiting the efflux pump functionality. In addition, the bacterial respiration rate, intracellular ATP level and tricarboxylic acid cycle (TCA) were promoted under the treatment of gramine. Notably, gramine effectively restored tigecycline activity in multiple animal infection models infected by tmexCD1-toprJ1 positive K. pneumoniae (RGF105-1). CONCLUSION: This study provides the first evidence of gramine's therapeutic potential as a novel tigecycline adjuvant for treating infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters.

Adaptive evolution of plasmid and chromosome contributes to the fitness of a blaNDM-bearing cointegrate plasmid in Escherichia coli.

Large cointegrate plasmids recruit genetic features of their parental plasmids and serve as important vectors in the spread of antibiotic resistance. They are now frequently found in clinical settings, raising the issue of how to limit their further transmission. Here, we conducted evolutionary research of a large blaNDM-positive cointegrate within Escherichia coli C600, and discovered that adaptive evolution of chromosome and plasmid jointly improved bacterial fitness, which was manifested as enhanced survival ability for in vivo and in vitro pairwise competition, biofilm formation, and gut colonization ability. From the plasmid aspect, large-scale DNA fragment loss is observed in an evolved clone. Although the evolved plasmid imposes a negligible fitness cost on host bacteria, its conjugation frequency is greatly reduced, and the deficiency of anti-SOS gene psiB is found responsible for the impaired horizontal transferability rather than the reduced fitness cost. These findings unveil an evolutionary strategy in which the plasmid horizontal transferability and fitness cost are balanced. From the chromosome perspective, all evolved clones exhibit parallel mutations in the transcriptional regulatory stringent starvation Protein A gene sspA. Through a sspA knockout mutant, transcriptome analysis, in vitro transcriptional activity assay, RT-qPCR, motility test, and scanning electron microscopy techniques, we demonstrated that the mutation in sspA reduces its transcriptional inhibitory capacity, thereby improving bacterial fitness, biofilm formation ability, and gut colonization ability by promoting bacterial flagella synthesis. These findings expand our knowledge of how cointegrate plasmids adapt to new bacterial hosts.

Vibration modes of three-dimensional spiral cochlea covering the organ of Corti.

So far, explaining the mechanism on active phonosensitive amplification in the cochlea is a major and difficult medical question. Among them, one of the key problems is that the motion pattern of the organ of Corti (OC) is still unknown. To this end, a multi-scale cochlear model including a three-dimensional spiral OC was established based on CT data and light source imaging experimental data, which complete combined the macroscopic and microscopic structure. On the basis of verifying the reliability of the model, acoustic-solid coupling calculation and modal analysis were performed on the model, and the vibration modes of basilar membrane (BM) and structures of the OC at different characteristic frequencies were discussed. The results show that tectorial membrane (TM) exhibits completely different vibration modes from BM at low frequencies, while the two movements gradually synchronize as the frequency increases. The amplitude position of OC's motion moves laterally with increasing frequency from Deiters' cells to Hensen's cells and then back to Deiters' cells. The OC exhibits longitudinal vibrations following BM when BM's displacement is large, while it manifests more as lateral movement of Deiters' cells when BM's displacement is small. This model can well simulate the motion process of BM and OC in the lymphatic fluid, which provides theoretical support and a numerical simulation computational platform to explore the interaction between macroscopic and microscopic tissue structures of the overall cochlea.

Phage-inspired strategies to combat antibacterial resistance.

Antimicrobial resistance (AMR) in clinically priority pathogensis now a major threat to public health worldwide. Phages are bacterial parasites that efficiently infect or kill specific strains and represent the most abundant biological entities on earth, showing great attraction as potential antibacterial therapeutics in combating AMR. This review provides a summary of phage-inspired strategies to combat AMR. We firstly cover the phage diversity, and then explain the biological principles of phage therapy that support the use of phages in the post-antimicrobial era. Furthermore, we state the versatility methods of phage therapy both from direct access as well as collateral access. Among the direct access approaches, we discuss the use of phage cocktail therapy, phage-encoded endolysins and the bioengineering for function improvement of used phages or endolysins. On the other hand, we introduce the collateral access, including the phages antimicrobial immunity combined therapy and phage-based novel antibacterial mimic molecules. Nowadays, more and more talented and enthusiastic scientist, doctors, pharmacists, media, authorities, and industry are promoting the progress of phage therapy, and proposed more phages-inspired strategy to make them more tractable to combat AMR and benefit more people, more animal and diverse environment in "one health" framework.

Pathogenic mechanism analysis of cochlear key structural lesion and phonosensitive hearing loss.

Due to ethical issues and the very fine and complex structure of the cochlea, it is difficult to directly perform experimental measurement on the human cochlea. Therefore, the finite element method has become an effective and replaceable new research means. Accurate numerical analysis on human ear using finite element method can provide better understanding of sound transmission and can be used to assess the influence of diseases on hearing and to treat hearing loss. In this research, a three-dimensional (3D) finite element model (FEM) of the human ear of cochlea was presented to investigate the destruction of basilar membrane (BM), round window (RW) sclerosis and perilymph fistula, the key structures of the cochlea, and analyze the effects of these abnormal pathological states in the cochlea on cochlear hearing, resulting in the changes in cochlear sense structure biomechanical behavior and quantitative prediction of the degree and harm of the disorder to the decline of human hearing. Therefore, this paper can deepen reader's understanding of the cochlear biomechanical mechanism and provide a theoretical foundation for clinical otology.

Coevolutionary phage training and Joint application delays the emergence of phage resistance in Pseudomonas aeruginosa.

Antibiotic-resistant bacteria are current threats to available antibiotic therapies, and this has renewed interest in the therapeutic use of phage as an alternative. However, development of phage resistance has led to unsuccessful therapeutic outcomes. In the current study, we applied phage training to minimize bacterial phage resistance and to improve treatment outcome by adapting the phage to their target hosts during co-evolution. We isolated and characterized a novel Pseudomonas aeruginosa N4-like lytic phage (PWJ) from wastewater in Yangzhou, China. PWJ is a double-stranded DNA podovirus that can efficiently lyse the model strain ATCC 27,853 and opportunistic pathogen PAO1. Genome sequencing of PWJ revealed features similar to those of the N4-like P. aeruginosa phage YH6. We used PWJ to screen for an evolved trained phage (WJ_Ev14) that restored infectivity to PWJ phage bacterial resisters. BLASTN analysis revealed that WJ_Ev14 is identical to its ancestor PWJ except for the amino acid substitution R1051S in its tail fiber protein. Moreover, phage adsorption tests and transmission electron microscopy of resistant bacteria demonstrated that the R1051S substitution was most likely the reason WJ_Ev14 could re-adsorb and regain infectivity. Furthermore, phage therapy assays in vitro and in a mouse P. aeruginosa lung infection model demonstrated that PWJ treatment resulted in improved clinical results and a reduction in lung bacterial load whereas the joint phage cocktail (PWJ+ WJ_Ev14) was better able to delay the emergence of resister bacteria. The phage cocktail (PWJ +WJ_Ev14) represents a promising candidate for inclusion in phage cocktails developed for clinical applications.

Long-read metagenomic sequencing reveals that high-copy small plasmids shape the highly prevalent antibiotic resistance genes in animal fecal microbiome.

The emergence and prevalence of animal-derived antibiotic resistance genes (ARGs) pose a great threat to public health globally. Long-read metagenomic sequencing is increasingly being used to decipher the fate of environmental ARGs. However, the investigations of the distribution, co-occurrence patterns, and host information of animal-derived environmental ARGs with long-read metagenomic sequencing have received little attention. To cover the gap, we employed a novel QitanTech nanopore long-read metagenomic sequencing method to perform a comprehensive and systematic investigation of the microbial communities and antibiotic resistance profiles, as well as to analyze the host information and genetic structures of ARGs in the feces of laying hens. Our results showed that highly abundant and diverse ARGs were detected in the feces of different ages of laying hens, indicating that feeding animal feces was an important reservoir for the enrichment and maintenance of ARGs. The distribution pattern of chromosomal ARGs was more strongly associated with fecal microbial communities than plasmid-mediated ARGs. Further long-read host tracking analysis revealed that ARGs from Proteobacteria are commonly located on plasmids, whereas in Firmicutes, they are usually carried by chromosomes. Co-occurrence analysis displayed that co-selection phenomena of different ARGs were common occurrences and highly active insertion sequences (ISs) could result in the serious prevalence of many ARGs. Notably, small high-copy plasmids played a significant role in the dissemination of several ARGs, such as floR and tet(L), which could disturb the compositions of fecal ARGs. Overall, our findings significantly expand our knowledge of the comprehensive landscape of feeding animal feces resistome, which is important for the prevention and management of multi-drug resistant bacteria in laying hens.

Gigantol restores the sensitivity of mcr carrying multidrug-resistant bacteria to colistin.

BACKGROUND: The emergence and wide spread of plasmid-mediated colistin resistance gene (mcr-1) and its mutants have immensely limited the efficacy of colistin in treating multidrug-resistant (MDR) Gram-negative bacterial infections. The development of synergistic combinations of antibiotics with a natural product that coped with the resistance of MDR bacteria was an economic strategy to restore antibiotics activity. Herein, we investigated gigantol, a bibenzyl phytocompound, for restoring in vitro and in vivo, the sensitivity of mcr-positive bacteria to colistin. METHODS: The synergistic activity of gigantol and colistin against multidrug-resistant Enterobacterales was studied via checkerboard assay and time-killing curve. Subsequently, the transcription and protein expression levels of mcr-1 gene were determined by RT-PCR and Western blots. The interaction of gigantol and MCR-1 was simulated via molecular docking and verified via site-directed mutagenesis of MCR-1. Hemolytic activity and cytotoxicity assay were used to evaluate the safety of gigantol. Finally, the in vivo synergistic effect was evaluated via two animal infection models. RESULTS: Gigantol restored the activity of colistin against mcr-positive bacteria E.coli B2 (MIC from 4 µg/ml to 0.25 µg/ml), Salmonella 15E343 (MIC from 8 µg/ml to 1 µg/ml), K. pneumoniae 19-2-1 (MIC from 32 µg/ml to 2 µg/ml) carrying mcr-1, mcr-3, mcr-8, respectively. Mechanistic studies revealed that gigantol down-regulated the expression of genes involved in LPS-modification, reduced the MCR-1 products and inhibited the activity of MCR-1 by binding to amino acid residues Tyr287 and Pro481 in its D-glucose-binding pocket. Safety evaluation showed that the addition of gigantol relieves the hemolysis caused by colistin. Compared with monotherapy, the combination of gigantol and colistin significantly improved the survival rate of Gallgallella mellonella larvae and mice infected by E.coli B2. Moreover, there was a considerable decrease in the bacterial load present in the viscera of mice. CONCLUSION: Our results confirmed that gigantol was a potential colistin adjuvant, and could be used to tackle multi-drug resistant Gram-negative pathogen infections combined with colistin.

Novel Trimethoprim Resistance Gene dfrA49 Identified in Riemerella anatipestifer from China.

Resistance to trimethoprim is mainly mediated by the acquisition of mobile dfrA genes, and most of them were discovered in Enterobacteriales. A total of 139 Riemerella anatipestifer isolates were collected from different farms in China during 2014 to 2020. Whole genome sequencing (WGS) and genome analysis of R. anatipestifer isolates revealed a 504-bp open reading frame (ORF) encoding a putative dfrA gene. This DfrA variant shared 66.47% amino acid sequence identity with DfrA36 and shared ≤51.20% identity with any other previously identified DfrA proteins. The novel dfrA gene, designated dfrA49, conferred trimethoprim (TMP) resistance when cloned into Escherichia coli BL21(DE3). Thirty dfrA49-positive isolates were identified from Jiangsu and Guangdong province (5/38, 13.16%, and 25/101, 24.75%, respectively). Five of the 38 isolates had obtained the complete genome sequences. Genomic analysis showed that the dfrA49 gene was located on chromosomes or a plasmid (four of them were on chromosomes and one was located on a plasmid). The plasmid p20190305E2-2_2 carried dfrA49, catB, ermF, ereD, blaOXA (88.36% identity with blaOXA-209), Δarr, and tet(X18). Further research indicated that dfrA49 usually coexisted with catB in R. anatipestifer. In this study, a novel trimethoprim resistance gene, dfrA49, was identified and characterized in chromosome and plasmid sequences from R. anatipestifer using WGS and bioinformatic methods. It further expands knowledge about the pool of mobile dfrA genes that confer resistance to trimethoprim and provides information about antibiotic resistance genes in R. anatipestifer, where the resistance gene pool circulating is not well understood. IMPORTANCE Trimethoprim is a synthetic antimicrobial agent inhibiting dihydrofolate reductase (DHFR), which is encoded by the folA gene. Acquired genes that confer trimethoprim resistance due to mutations in the folA gene are designated dfr and divided into two main families including dfrA and dfrB. Resistance to trimethoprim is mainly mediated by the acquisition of mobile dfrA genes, and most of them were discovered in Enterobacteriales. R. anatipestifer belongs to the Flavobacteriaceae family, and the reservoir of dfrA resistance genes in R. anatipestifer has not been fully investigated. A novel trimethoprim resistance gene, dfrA49, which was identified and characterized in chromosome and plasmid sequences in this study, increased the MIC of TMP (>256-fold) in E. coli BL21(DE3). Our study expands knowledge about the pool of mobile dfrA genes that confer resistance to trimethoprim and broadens the understanding of the host spectrum of dfrA family genes.

Enterobacteriaceae genome-wide analysis reveals roles for P1-like phage-plasmids in transmission of mcr-1, tetX4 and other antibiotic resistance genes.

P1 -like phage-plasmids (PPs) are important gene vehicles in isolated pathogens. In this study, we conducted genome-wide and cross-species analysis of antimicrobial resistance genes (ARGs) from 35 ARG-positive P1-like PPs. LS-BSR analysis reveal that P1-like PPs had in common 7 highly variable regions and carried 48 different ARG subtypes. The most prevalent gene groups were the colistin resistance gene mcr-1 and a class 1 integron. Analysis of the flanking sequences of mcr-1 indicated an "IS30-mcr-1-ORF-IS30" as the core cluster. In particular, we found an mcr-1- and blaCTX-M-55-coharboring large fusion P1-like PP. Also, tet(X4) was detected and flanking sequences indicated tet(X4)-bearing cluster can formed a larger size fusion plasmid mediated a wider spread via IS26 hotspots. Overall, this study demonstrated that P1-like PPs can not only mobilize a large number of ARGs in variable regions but also form larger hybrid P1-like PPs that would increase their ability to spread antimicrobial resistance.

Development of an Assessment Model for the Effect of the Replacement of Minimal Artificial Ossicles on Hearing in the Inner Ear.

Due to ethical issues and the nature of the ear, it is difficult to directly perform experimental measurements on living body elements of the human ear. Therefore, a numerical model has been developed to effectively assess the effect of the replacement of artificial ossicles on hearing in the inner ear. A healthy volunteer's right ear was scanned to obtain CT data, which were digitalized through the use of a self-compiling program and coalescent Patran-Nastran software to establish a 3D numerical model of the whole ear, and a frequency response of a healthy human ear was analyzed. The vibration characteristics of the basilar membrane (BM) after total ossicular replacement prosthesis (TORP) implantation were then analyzed. The results show that although the sound conduction function of the middle ear was restored after replacement of the TORP, the sensory sound function of the inner ear was affected. In the low frequency and medium frequency range, hearing loss was 5.2~10.7%. Meanwhile, in the middle-high frequency range, the replacement of a middle ear TORP in response to high sound pressure produced a high acoustic stimulation effect in the inner ear, making the inner ear structures susceptible to fatigue and more prone to fatigue damage compared to the structures in healthy individuals. This developed model is able to assess the effects of surgical operation on the entire hearing system.

Genomic and functional analysis of high-level tigecycline resistant Klebsiella michiganensis co-carrying tet(X4) and tmexCD2-toprJ2 from pork.

Tigecycline plays an important role in the clinical treatment of infections caused by multidrug-resistant pathogens. The emergence of plasmid-mediated tigecycline resistance genes tet(X) and tmexCD1-tmexJ1 has been reported in a variety of animal and animal-derived foods, and have the potential spread to humans, seriously limiting the choice of clinical medication. Herein, three ST92 Klebsiella michiganensis isolates co-harboring tet(X4) and tmexCD2-toprJ2 were collected from pork samples in Jiangsu Province, China. These K. michiganensis isolates were all multidrug-resistant isolates. Genome analysis showed that tmexCD2-toprJ2 and tet(X4) were located on IncFIB(K) and IncX1 plasmids, respectively. The IncFIB(K) plasmid pMX581-77k is a novel tmexCD2-toprJ2-bearing plasmid. Worryingly, there were only a small number of SNPs between K. michiganensis isolated from pork in this study and K. michiganensis from human sources, with the possibility of clonal transmission. In addition, tet(X4) and tmexCD2-toprJ2 in K. michiganensis were able to stabilize in the absence of antibiotics. The growth curve indicated that the tmexCD2-toprJ2-positive plasmid imposed a burden on the growth of host bacteria. Interestingly, we found that the high-level resistance phenotype to tigecycline in these K. michiganensis isolates was mainly mediated by tet(X4). However, both tet(X4) and tmexCD2-toprJ2 expression were significantly elevated when host bacteria were exposed to tigecycline. This study systematically investigated K. michiganensis co-carrying tet(X4) and tmexCD2-toprJ2, emphasizing the importance for continuous surveillance of tigecycline-resistant K. michiganensis in animal-derived foods.

The Prevalence of Plasmid-Mediated Colistin Resistance Gene mcr-1 and Different Transferability and Fitness of mcr-1-Bearing IncX4 Plasmids in Escherichia coli from Pigeons.

The prevalence of colistin-resistant bacteria limited the usage of colistin in the treatment of clinical multidrug-resistant Gram-negative bacterial infections. Here, we aimed to investigate the prevalence and molecular characterization of mcr-1-carrying isolates from pigeons close to humans following the ban on the use of colistin as an animal feed additive in China. Methods, including PCR, antimicrobial susceptibility testing, conjugation experiments, plasmid replicon typing, genome sequencing, bioinformatics analysis, measurement of growth curves, competition experiments, and plasmid stability assays were used to identify and characterize mcr-1-positive isolates. In total, 45 mcr-1-positive E. coli isolates were acquired from 100 fecal samples, and MICs of colistin ranged from 4 to 8 mg/L. The prevalence of mcr-1-positive E. coli isolates from pigeons was mainly mediated by IncX4 plasmids (39/45), including transferable mcr-1-bearing IncX4 plasmids with fitness advantage in 21 isolates, and nontransferable mcr-1-bearing IncX4 plasmids with fitness disadvantage in 18 isolates. There is a similar structure among the 6 mcr-1-bearing nontransferable IncX4 plasmids and 10 mcr-1-bearing transferable IncX4 plasmids in 16 E. coli isolates that have been sequenced. Plasmid transferability evaluation indicated that the same IncX4 plasmid has different transferability in different E. coli isolates. In conclusion, this study demonstrates that pigeons could act as potential reservoirs for the spread of mcr-1-positive E. coli in China. Transferability of IncX4 plasmids may be influenced by host chromosome in the same bacterial species. Additional research on the factors influencing the transferability of IncX4 plasmids in different bacterial hosts is required to help combat antimicrobial resistance. IMPORTANCE The emergence of plasmid-mediated colistin resistance gene mcr-1 incurs great concerns. Since the close proximity of pigeons with humans, it is significant to understand the prevalence and molecular characterization of mcr-1-positive isolates in pigeons, to provide a rationale for controlling its spread. Here, we found that the prevalence of mcr-1-positive E. coli from pigeons was mainly mediated by IncX4 plasmids. However, different transferability and fitness of mcr-1-bearing IncX4 plasmids in E. coli were observed, which demonstrated that transferability of IncX4 plasmids could be affected not only by genes on plasmids, but also by chromosomal factors in the same bacterial species. Our finding provided a new insight on studying the factors influencing the transferability of plasmids.

Uncovering the determinants of model Escherichia coli strain C600 susceptibility and resistance to lytic T4-like and T7-like phage.

As antimicrobial resistance (AMR) continues to increase, the therapeutic use of phages has re-emerged as an attractive alternative. However, knowledge of phage resistance development and bacterium-phage interaction complexity are still not fully interpreted. In this study, two lytic T4-like and T7-like phage infecting model Escherichia coli strain C600 are selected, and host genetic determinants involved in phage susceptibility and resistance are also identified using TraDIS strategy. Isolation and identification of the lytic T7-like show that though it belongs to the phage T7 family, genes encoding replication and transcription protein exhibit high differences. The TraDIS results identify a huge number of previously unidentified genes involved in phage infection, and a subset (six in susceptibility and nine in resistance) are shared under pressure of the two kinds of lytic phage. Susceptible gene wbbL has the highest value and implies the important role in phage susceptibility. Importantly, two susceptible genes QseE (QseE/QseF) and RstB (RstB/RstA), encoding the similar two-component system sensor histidine kinase (HKs), also identified. Conversely and strangely, outer membrane protein gene ompW, unlike the gene ompC encoding receptor protein of T4 phage, was shown to provide phage resistance. Overall, this study exploited a genome-wide fitness assay to uncover susceptibility and resistant genes, even the shared genes, important for the E. coli strain of both most popular high lytic T4-like and T7-like phages. This knowledge of the genetic determinants can be further used to analysis the behind function signatures to screen the potential agents to aid phage killing of MDR pathogens, which will greatly be valuable in improving the phage therapy outcome in fighting with microbial resistance.

Similarities of P1-Like Phage Plasmids and Their Role in the Dissemination of blaCTX-M-55.

The P1-like phage plasmid (PP) has been widely used as a molecular biology tool, but its role as an active accessory cargo element is not fully understood. In this study, we provide insights into the structural features and gene content similarities of 77 P1-like PPs in the RefSeq database. We also describe a P1-like PP carrying a blaCTX-M-55 gene, JL22, which was isolated from a clinical strain of Escherichia coli from a duck farm. P1-like PPs were very similar and conserved based on gene content similarities, with only eight highly variable regions. Importantly, two kinds of replicon types, namely, IncY and p0111, were identified and can be used to specifically identify the P1-like phage. JL22 is similar to P1, acquiring an important foreign DNA fragment with two obvious features, namely, the plasmid replication gene repA' (p0111) replacing the gene repA (IncY) and a 4,200-bp fragment mobilized by IS1380 and IS5 and containing a blaCTX-M-55 gene and a trpB gene encoding tryptophan synthase (indole salvaging). The JL22 phage could be induced but had no lytic capacities. However, a lysogenic recipient and intact structure of JL22 virions were observed, showing that the extended-spectrum ß-lactamase blaCTX-M-55 gene was successfully transferred. Overall, conserved genes can be a good complement to improve the identification efficiency and accuracy in future screening for P1-like PPs. Moreover, the highly conserved structures may be important for their prevalence and dissemination. IMPORTANCE As a PP, P1 DNA exists as a low-copy-number plasmid and replicates autonomously with a lysogenization style. This unique mode of P1-like elements probably indicates a stable contribution to antibiotic resistance. After analyzing these elements, we show that P1-like PPs are very similar and conserved, with only eight highly variable regions. Moreover, we observed the occurrence of replicon IncY and p0111 only in the P1-like PP community, implying that these conserved regions, coupled with IncY and p0111, can be an important complement in future screening of P1-like PPs. Identification and characterization of JL22 confirmed our findings that major changes were located in variable regions, including the first detection of blaCTX-M-55 in such a mobile genetic element. This suggests that these variable regions may facilitate foreign DNA mobilization. This study features a comprehensive genetic analysis of P1-like PPs, providing new insights into the dissemination mechanisms of antibiotic resistance through P1 PPs.

Molecular mechanisms and genomic basis of tigecycline-resistant Enterobacterales from swine slaughterhouses.

The continuous emergence of tigecycline-resistant bacteria is undermining the effectiveness of clinical tigecycline. Environmental tigecycline-resistant bacteria have the potential to infect humans through human-environment interactions. Furthermore, the mechanisms of tigecycline resistance in Enterobacterales are complicated. In this study, we aimed to investigate the additional pathways of tigecycline resistance in environmental Enterobacterales besides tet(X) and tmexCD-toprJ. During the years 2019-2020, tigecycline-resistant Enterobacterales (n = 45) negative for tet(X) and tmexCD-toprJ were recovered from 328 different samples from two slaughterhouses. Five distinct bacteria species were identified, of which Klebsiella pneumoniae (n = 37) was the most common, with K. pneumoniae ST45 and ST35 being the predominant clones. Tigecycline resistance determinants analysis showed that tet(A) mutations and ramR inactivation were the most prevalent mechanisms for tigecycline resistance in the 45 strains. Two known tet(A) variants (type 1 and tet(A)-v) and one novel tet(A) variant (type 3) were identified. Cloning experiments confirmed that the novel type 3 tet(A) could enhance the 4-fold MIC for tigecycline. Inactivation of ramR was induced by either point mutations or indels of sequences, which could result in the overexpression of AcrAB pump genes leading to tigecycline resistance. In addition, all isolates were resistant to a wide range of antimicrobials and carried various resistance genes. These findings enriched the epidemiological and genomic characterizations of tigecycline-resistant Enterobacterales from slaughterhouses and contributed to a better understanding of the complex mechanisms of tigecycline resistance in environmental bacteria.

Genetic Diversity Analysis of Sapindus in China and Extraction of a Core Germplasm Collection Using EST-SSR Markers.

Sapindus is an important forest tree genus with utilization in biodiesel, biomedicine, and it harbors great potential for biochemical engineering applications. For advanced breeding of Sapindus, it is necessary to evaluate the genetic diversity and construct a rationally designed core germplasm collection. In this study, the genetic diversity and population structure of Sapindus were conducted with 18 expressed sequence tag-simple sequence repeat (EST-SSR) markers in order to establish a core germplasm collection from 161 Sapindus accessions. The population of Sapindus showed high genetic diversity and significant population structure. Interspecific genetic variation was significantly higher than intraspecific variation in the Sapindus mukorossi, Sapindus delavayi, and combined Sapindus rarak plus Sapindus rarak var. velutinus populations. S. mukorossi had abundant genetic variation and showed a specific pattern of geographical variation, whereas S. delavayi, S. rarak, and S. rarak var. velutinus showed less intraspecific variation. A core germplasm collection was created that contained 40% of genetic variation in the initial population, comprising 53 S. mukorossi and nine S. delavayi lineages, as well as single representatives of S. rarak and S. rarak var. velutinus. These results provide a germplasm basis and theoretical rationale for the efficient management, conservation, and utilization of Sapindus, as well as genetic resources for joint genomics research in the future.

Acylated homoserine lactones regulate the response of methane metabolism and nitrogen metabolism to florfenicol in anaerobic fermentation.

Antimicrobial agents enter the ecological environment through animal excreta and disrupt metabolism in environmental microorganisms. Quorum sensing (QS) can help bacteria adapt to their surroundings. To explore how acyl-homoserine lactone (AHL) can adjust the influence of florfenicol on nitrogen cycling and methane metabolism in anaerobic fermentation, a small indoor thermostatic anaerobic fermentation model was established by adding exogenous acylated homoserine lactone (AHL) signal molecules with florfenicol as the stress factor. Through bacterial function prediction by PICRUST, we found that the addition of AHL further increased the promotion of methanogenesis_by_CO2_reduction_with_H2 and hydrogenotrophic methanogenesis by florfenicol. Before the third sampling, florfenicol significantly inhibited the enrichment of the denitrification pathway microbiota, whereas the addition of AHL significantly promoted the enrichment of the denitrification pathway microbiota. Functional annotation showed that florfenicol exposure stress significantly affected nitrogen and methane metabolism, and the addition of AHLs reduced the response of functional genes to florfenicol. All nitrogen cycling enzymes with significantly different abundances in treatment groups were substantially associated with methane-metabolizing enzymes. Glutamate metabolism is significant in the process of anaerobic fermentation, and is a correlation point between nitrogen and methane metabolism. In our experiment, AHL was the influencing factor at the highest latitude that directly regulates the metabolism of NO3--N and the degradation process of florfenicol. The addition of AHL curbed the inhibitory effect of florfenicol on some functional microbiota, improved the stability of fermentation microbiota, and weakened the impact of antibiotic residues by improving its degradation efficiency.

Phenotypic and genomic analysis reveals Riemerella anatipestifer as the potential reservoir of tet(X) variants.

BACKGROUND: Tigecycline is regarded as one of the last-resort antimicrobials clinically. Emergence of plasmid-mediated tet(X) undermines such an important drug. However, the origins of tet(X) remain largely unexplored. METHODS: Riemerella anatipestifer strains were characterized by PCR, antimicrobial susceptibility testing, WGS and bioinformatics analysis. Functional analysis of tet(X) was verified by cloning experiments. Genomic structures of chromosome- and plasmid-mediated tet(X) were analysed. RESULTS: Thirty-eight R. anatipestifer strains were collected and found to be positive for tet(X). These strains were resistant to multiple antimicrobials; 55.3% (21/38) of the strains were resistant to tigecycline and all of the strains demonstrated resistance to tetracycline. The complete genome sequences of 18 representative strains were obtained. WGS analysis of 38 genomes identified 13 tet(X) variants located on chromosomes, which increased MICs of tigecycline (16-256-fold) for Escherichia coli, although most of them could not confer high-level resistance to tigecycline in the original R. anatipestifer hosts. Genomic environment analysis indicated that the occurrence of multiple tet(X) variants is common and other resistance genes, such as catB, tet(Q), floR, blaOXA, ereD and ermF, could be located in the same chromosomal regions. Two types of tet(X)-bearing segments were identified, one of which was floR-ISCR2-tet(X). This indicates that tet(X) variants were not conserved in chromosomal structures, but in regions with potential transferability. Furthermore, an MDR plasmid carrying tet(X18) was found in R. anatipestifer 20190305E2-2, different from the chromosomal tet(X21). CONCLUSIONS: This study confirmed that tet(X) is highly prevalent in R. anatipestifer. The transfer risk of tet(X) across R. anatipestifer to other clinical pathogens warrants further investigations.

Antimicrobial Resistance and Genomic Epidemiology of tet(X4)-Bearing Bacteria of Pork Origin in Jiangsu, China.

The emergence of tigecycline-resistant bacteria in agri-food chains poses a public health concern. Recently, plasmid-mediated tet(X4) was found to be resistant to tigecycline. However, genome differences between tet(X4)-positive Escherichia coli of human and pork origins are still under-investigated. In this study, 53 pork samples were collected from markets in Jiangsu, China, and 23 tet(X4)-positive isolates were identified and shown to confer resistance to multiple antibiotics, including tigecycline. tet(X4)-positive isolates were mainly distributed in E. coli (n = 22), followed by Klebsiella pneumoniae (n = 1). More than half of the tet(X4) genes were able to be successfully transferred into E. coli C600. We downloaded all tet(X4)-positive E. coli isolates from humans and pork found in China from the NCBI database. A total of 42 known STs were identified, of which ST10 was the dominant ST. The number of ARGs and plasmid replicons carried by E. coli of human origin were not significantly different from those carried by E. coli of pork origin. However, the numbers of insertion sequences and virulence genes carried by E. coli of human origin were significantly higher than those carried by E. coli of pork origin. In addition to E. coli, we analyzed all 23 tet(X4)-positive K. pneumoniae strains currently reported. We found that these tet(X4)-positive K. pneumoniae were mainly distributed in China and had no dominant STs. This study systematically investigated the tet(X4)-positive isolates, emphasizing the importance of the continuous surveillance of tet(X4) in pork.