Bavachin Rejuvenates Sensitivity of Colistin against Colistin-Resistant Gram-Negative Bacteria.

The emergence of plasmid-mediated colistin resistance threatens the efficacy of colistin as a last-resort antibiotic used to treat infection caused by Gram-negative bacteria (GNB). Given the shortage of new antibiotics, the discovery of adjuvants to existing antibiotics is a promising strategy to combat infections caused by multidrug-resistant (MDR) GNB. This study was designed to investigate the potential synergistic antibacterial activity of bavachin, a bioactive compound extracted from the Psoralea Fructus, combined with colistin against MDR GNB. Herein, the synergistic efficacy in vitro and the therapeutic efficacy of colistin combined with bavachin in vivo were evaluated. The synergistic mechanism was detected by fluorescent probe and the transcript levels of mcr-1. Bavachin combined with colistin showed an excellent synergistic activity against GNB, as the FICI ≤ 0.5. In contrast to colistin alone, combination therapy dramatically increased the survival rate of Galleria mellonella and mice in vivo. Moreover, the combination of bavachin and colistin significantly reduced the amount of bacterial biofilm formation, improved the membrane disruption of colistin and inhibited mcr-1 transcription. These findings show that bavachin is a potential adjuvant of colistin, which may provide a new strategy to combat colistin-resistant bacteria infection with lower doses of colistin.

Prevalence and genetic characteristics of fosB-positive Staphylococcus aureus in duck farms in Guangdong, China in 2020.

OBJECTIVES: To investigate the epidemiology of fosB-positive Staphylococcus aureus in waterfowl farms in the Pearl River tributaries in Guangdong Province, China in 2020. METHODS: A total of 63 S. aureus were recovered from 315 samples collected from six duck farms and one goose farm. PFGE, WGS and analysis were performed on 19 fosB-positive S. aureus. RESULTS: The fosfomycin resistance rate of the strains was as high as 52.4% (33/63), and 30.1% (19/63) of the strains carried fosB. Resistance gene prediction results showed that duck farm environment-derived strains contained the oxazolidinone drug resistance gene optrA. All fosB-positive S. aureus were MRSA and most of them were MDR, mainly ST9-t899 and ST164-t899. PFGE showed that fosB-positive S. aureus from humans and ducks could be clustered into the same clade. In addition, core-genome SNP analysis showed that clonal transmission of S. aureus occurred between humans and water. Pan-genome analysis showed that S. aureus had an open pangenome. The fosB gene was located on 2610-2615 bp plasmids, which all contained a broad host-range plasmid replication protein family 13. Small plasmids carrying the fosB gene could be found in different multilocus STs of S. aureus. CONCLUSIONS: This study indicated that duck farms in Guangdong, China could be an important reservoir of fosB-positive S. aureus. The spread of drug-resistant bacteria in waterfowl farms requires further monitoring.

Metagenomic analysis of antimicrobial resistance in ducks, workers, and the environment in duck farms, southern China.

Duck farms are one of the important reservoirs of antimicrobial resistance genes (ARGs) that spread to humans and the environment. However, few studies have focused on the characteristics of antimicrobial profiles in duck farms. Here we explored the distribution characteristics and potential transmission mechanisms of ARGs in ducks, farm workers, and the environment in duck farms by a metagenomic approach. The results showed that the highest abundance and diversity of ARGs were found in duck manure. The abundance and diversity of ARGs in workers and environmental samples were higher than those in the control group. tet(X) and its variants were prevalent in duck farms, with tet(X10) being the most abundant. The genetic structure "tet(X)-like + α/ß hydrolase" was found in ducks, workers, and the environment, implying that tet(X) and its variants have been widely spread in duck farms. Network analysis indicated that ISVsa3 and IS5075 might play an important role in the coexistence of ARGs and metal resistance genes (MRGs). The Mantel tests showed that mobile genetic elements (MGEs) were significantly correlated with ARG profiles. The results suggest that duck manure may be a potential hotspot source of ARGs, including tet(X) variants that spread to the surrounding environment and workers via MGEs. These results help us optimize the antimicrobials strategy and deepen our understanding of ARG spread in duck farms.

Evaluating the Antibacterial and Antivirulence Activities of Floxuridine against Streptococcus suis.

Streptococcus suis is an emerging zoonotic pathogen that can cause fatal diseases such as meningitis and sepsis in pigs and human beings. The overuse of antibiotics is leading to an increased level of resistance in S. suis, and novel antimicrobial agents or anti-virulence agents for the treatment of infections caused by S. suis are urgently needed. In the present study, we investigated the antibacterial activity, mode of action and anti-virulence effects of floxuridine against S. suis. Floxuridine showed excessive antibacterial activity against S. suis both in vivo and in vitro; 4 × MIC of floxuridine could kill S. suis within 8 h in a time-kill assay. Meanwhile, floxuridine disrupted the membrane structure and permeability of the cytoplasmic membrane. Molecular docking revealed that floxuridine and SLY can be directly bind to each other. Moreover, floxuridine effectively inhibited the hemolytic capacity and expression levels of the virulence-related genes of S. suis. Collectively, these results indicate that the FDA-approved anticancer drug floxuridine is a promising agent and a potential virulence inhibitor against S. suis.

Isopropoxy Benzene Guanidine Ameliorates Streptococcus suis Infection In Vivo and In Vitro.

Streptococcus suis, an encapsulated zoonotic pathogen, has been reported to cause a variety of infectious diseases, such as meningitis and streptococcal-toxic-shock-like syndrome. Increasing antimicrobial resistance has triggered the need for new treatments. In the present study, we found that isopropoxy benzene guanidine (IBG) significantly attenuated the effects caused by S. suis infection, in vivo and in vitro, by killing S. suis and reducing S. suis pathogenicity. Further studies showed that IBG disrupted the integrity of S. suis cell membranes and increased the permeability of S. suis cell membranes, leading to an imbalance in proton motive force and the accumulation of intracellular ATP. Meanwhile, IBG antagonized the hemolysis activity of suilysin and decreased the expression of Sly gene. In vivo, IBG improved the viability of S. suis SS3-infected mice by reducing tissue bacterial load. In conclusion, IBG is a promising compound for the treatment of S. suis infections, given its antibacterial and anti-hemolysis activity.

Preliminary view of the global distribution and spread of the tet(X) family of tigecycline resistance genes.

BACKGROUND: The emergence of plasmid-mediated tet(X3)/tet(X4) genes is threatening the role of tigecycline as a last-resort antibiotic to treat clinical infections caused by XDR bacteria. Considering the possible public health threat posed by tet(X) and its variants [which we collectively call 'tet(X) genes' in this study], global monitoring and surveillance are urgently required. OBJECTIVES: Here we conducted a worldwide survey of the global distribution and spread of tet(X) genes. METHODS: We analysed a comprehensive dataset of bacterial genomes in conjunction with surveillance data from our laboratory and the NCBI database, as well as sufficient metadata to characterize the results. RESULTS: The global distribution features of tet(X) genes were revealed. We clustered three types of genetic backbones of tet(X) genes embedded or transferred in bacterial genomes. Our pan-genome analyses revealed a large genetic pool composed of tet(X)-carrying sequences. Moreover, phylogenetic trees of tet(X) genes and tet(X)-like proteins were built. CONCLUSIONS: To the best of our knowledge, our results provide the first view of the global distribution of tet(X) genes, demonstrate the features of tet(X)-carrying fragments and highlight the possible evolution of tigecycline-inactivation enzymes in diverse bacterial species and habitats.

Application of manure containing tetracyclines slowed down the dissipation of tet resistance genes and caused changes in the composition of soil bacteria.

Manure application contributes to the increased environmental burden of antibiotic resistance genes (ARGs). We investigated the response of tetracycline (tet) resistance genes and bacterial taxa to manure application amended with tetracyclines over two months. Representative tetracyclines (oxytetracycline, chlorotetracycline and doxycycline), tet resistance genes (tet(M), tet(O), tet(W), tet(S), tet(Q) and tet(X)) and bacterial taxa in the untreated soil, +manure, and +manure+tetracyclines groups were analyzed. The abundances of all tet resistance genes in the +manure group were significantly higher than those in the untreated soil group on day 1. The abundances of all tet resistance genes (except tet(Q) and tet(X)) were significantly lower in the +manure group than those in the +manure+tetracyclines group on day 30 and 60. The dissipation rates were higher in the +manure group than those in the +manure+tetracyclines group. Disturbance of soil bacterial community composition imposed by tetracyclines was also observed. The results indicated that tetracyclines slowed down the dissipation of tet resistance genes in arable soil after manure application. Application of manure amended with tetracyclines may provide a significant selective advantage for species affiliated to the taxonomical families of Micromonosporaceae, Propionibacteriaceae, Streptomycetaceae, Nitrospiraceae and Clostridiaceae.

Fate of potential indicator antimicrobial resistance genes (ARGs) and bacterial community diversity in simulated manure-soil microcosms.

The aim of this study was to investigate the fate of nine potential indicator antimicrobial resistance genes (ARGs) (sul1, sul2, tetB, tetM, ermB, ermF, fexA, cfr, intI1) and the diversity of bacterial communities in response to poultry manure applications to arable soil over a 90 day period. Quantitative real time PCR and Illumina high-throughput sequencing of 16S rDNA gene were used to quantify and trace ARG fate. The levels of all genes dramatically decreased over time and intI1, sul1, sul2 and tetM always had the greatest abundance and lowest dissipation rates. This indicated that more effort should be focused on the ARG elimination from manure rather than waiting for subsequent attenuation in the environment. Our sequencing results documented dramatic changes in the microbial community structure and diversity during these experiments. In poultry manure groups, Bacteroidetes and Actinobacteria were the two dominant phyla while Acidobacteria dominated the control groups. Moreover, the relative abundance of genera Corynebacterium, Pseudomonas, Ochrobactrum, Actinomadura and Bacillus, which contained potential opportunistic pathogens, changed over time suggesting that poultry manure not only strongly influenced bacterial community composition, but also selected specific bacterial communities. This study provides a glimpse of ARG fates and bacterial community diversity in soil after the application of poultry manure.

Metagenomic insights into the effect of oxytetracycline on microbial structures, functions and functional genes in sediment denitrification.

Denitrification is an indispensable pathway of nitrogen removal in aquatic ecosystems, and plays an important role in decreasing eutrophication induced by excessive reactive nitrogen pollution. Aquatic environments also suffer from antibiotic pollution due to runoff from farms and sewage systems. The aim of this study was to investigate the effect of oxytetracycline stress on denitrifying functional genes, the microbial community and metabolic pathways in sediments using high-throughput sequencing and metagenomic analysis. The oxytetracycline was observed to significantly inhibit the abundance of nirK and nosZ genes (P < 0.001). KEGG pathway annotation indicated that oxytetracycline treatment decreased the abundance of nitrate reductase, nitrite reductase and N2O reductase. Functional annotations revealed that oxytetracycline exposure decreased the abundance of the protein metabolism subsystem in the bacterial community. Metagenomic sequencing demonstrated that the abundance of Proteobacteria and Firmicutes increased with oxytetracycline exposure while the Actinobacteria decreased. In sediments, Pseudomonas and Bradyrhizobium were major contributors to denitrification and oxytetracycline exposure resulted in a decreased abundance of Bradyrhizobium. These results indicated that oxytetracycline residues influences the denitrifier community and may heighten occurrence of reactive nitrogen in aquatic ecosystems.

Bioprospecting for ß-lactam resistance genes using a metagenomics-guided strategy.

Emergence of new antibiotic resistance bacteria poses a serious threat to human health, which is largely attributed to the evolution and spread of antibiotic resistance genes (ARGs). In this work, a metagenomics-guided strategy consisting of metagenomic analysis and function validation was proposed for rapidly identifying novel ARGs from hot spots of ARG dissemination, such as wastewater treatment plants (WWTPs) and animal feces. We used an antibiotic resistance gene database to annotate 76 putative ß-lactam resistance genes from the metagenomes of sludge and chicken feces. Among these 76 candidate genes, 25 target genes that shared 40~70% amino acid identity to known ß-lactamases were cloned by PCR from the metagenomes. Their resistances to four ß-lactam antibiotics were further demonstrated. Furthermore, the validated ARGs were used as the reference sequences to identify novel ARGs in eight environmental samples, suggesting the necessity of re-examining the profiles of ARGs in environmental samples using the validated novel ARG sequences. This metagenomics-guided pipeline does not rely on the activity of ARGs during the initial screening process and may specifically select novel ARG sequences for function validation, which make it suitable for the high-throughput screening of novel ARGs from environmental metagenomes.

Antibiotics, Antibiotic Resistance Genes, and Bacterial Community Composition in Fresh Water Aquaculture Environment in China.

Environmental antibiotic resistance has drawn increasing attention due to its great threat to human health. In this study, we investigated concentrations of antibiotics (tetracyclines, sulfonamides and (fluoro)quinolones) and abundances of antibiotic resistance genes (ARGs), including tetracycline resistance genes, sulfonamide resistance genes, and plasmid-mediated quinolone resistance genes, and analyzed bacterial community composition in aquaculture environment in Guangdong, China. The concentrations of sulfametoxydiazine, sulfamethazine, sulfamethoxazole, oxytetracycline, chlorotetracycline, doxycycline, ciprofloxacin, norfloxacin, and enrofloxacin were as high as 446 µg kg(-1) and 98.6 ng L(-1) in sediment and water samples, respectively. The relative abundances (ARG copies/16S ribosomal RNA (rRNA) gene copies) of ARGs (sul1, sul2, sul3, tetM, tetO, tetW, tetS, tetQ, tetX, tetB/P, qepA, oqxA, oqxB, aac(6')-Ib, and qnrS) were as high as 2.8 × 10(-2). The dominant phyla were Proteobacteria, Bacteroidetes, and Firmicutes in sediment samples and Proteobacteria, Actinobacteria and Bacteroidetes in water samples. The genera associated with pathogens were also observed, such as Acinetobacter, Arcobacter, and Clostridium. This study comprehensively investigated antibiotics, ARGs, and bacterial community composition in aquaculture environment in China. The results indicated that fish ponds are reservoirs of ARGs and the presence of potential resistant and pathogen-associated taxonomic groups in fish ponds might imply the potential risk to human health.

Fate of metal resistance genes in arable soil after manure application in a microcosm study.

Manure application contributes to the spread and persistence of metal resistance genes (MRGs) in the environment. We investigated the fate of copper (Cu) and zinc (Zn) resistance genes (pcoA, pcoD and zntA) in arable soil after Cu/Zn-containing manure application. Manure with or without addition of metals (Cu/Zn) was added in a soil microcosm over 2 months. Soil samples were collected for analysis on day 0, 30 and 60. The abundances of all MRGs (pcoA, pcoD and zntA) in manure group were significantly higher than those in untreated soil and manure+metals groups. All MRGs dissipated 1.2-1.3 times faster in manure group (from -90 ± 8% to -93 ± 7%) than those in manure+metals group (from -68 ± 8% to -78 ± 5%). The results indicated that manure from healthy pigs contributed to the occurrence of metals (Cu/Zn) and MRGs (pcoA, pcoD and zntA) in arable soil. The significant effects of manure application on the accumulation of pcoA, pcoD and zntA lasted for 1-2 months. Cu/Zn can slow down the dissipation of pcoA, pcoD and zntA after manure application. This is the first report to investigate the fate of MRGs in soil after manure application.

The synergistic antibacterial activity and mechanism of colistin-oxethazaine combination against gram-negative pathogens.

Background: The rapid spread of bacteria with plasmid-mediated resistance to antibiotics poses a serious threat to public health. The search for potential compounds that can increase the antibacterial activity of existing antibiotics is a promising strategy for addressing this issue. Methods: Synergistic activity of the FDA-approved agent oxethazine combined with colistin was investigated in vitro using checkerboard assays and time-kill curves. The synergistic mechanisms of their combination of oxethazine and colistin was explored by fluorescent dye, scanning electron microscopy (SEM) and LC-MS/MS. The synergistic efficacy was evaluated in vivo by the Galleria mellonella and mouse sepsis models. Results: In this study, we found that oxethazine could effectively enhance the antibacterial activity of colistin against both mcr-positive and -negative pathogens, and mechanistic assays revealed that oxethazine could improve the ability of colistin to destruct bacterial outer membrane and cytoplasmic membrane permeability. In addition, their combination triggered the accumulation of reactive oxygen species causing additional damage to the membrane structure resulting in cell death. Furthermore, oxethazine significantly enhanced the therapeutic efficacy of colistin in two animal models. Conclusion: These results suggested that oxethazine, as a promising antibiotic adjuvant, can effectively enhance colistin activity, providing a potential strategy for treating multidrug-resistant bacteria.

First insights into the prevalence, genetic characteristics, and pathogenicity of Bacillus cereus from generations worldwide.

Bacillus cereus, a global threat, is one of the major causes of toxin-induced foodborne diseases. However, a comprehensive assessment of the prevalence and characteristics of B. cereus worldwide is still lacking. Here, we applied whole-genome sequence analysis to 191 B. cereus collected in Africa, America, Asia, Europe, and Oceania from the 1900s to 2022, finding that CC142 dominated the global B. cereus clonal complex. The results provided direct evidence that B. cereus could spread through the food chain and intercontinentally. B. cereus from different generations worldwide showed coherence in the antibiotic-resistant gene and virulence and biofilm gene profiles, although with high genomic heterogeneity. The BCI-BCII-vanZF-fosB profiles and virulence and biofilm genes were detected at high rates, and we emphasized that B. cereus would pose a serious challenge to global public health and clinical treatment.IMPORTANCEThis study first emphasized the prevalence, genetic characteristics, and pathogenicity of Bacillus cereus worldwide from the 1900s to 2022 using whole-genome sequence analysis. The CC142 dominated the global Bacillus cereus clonal complex. Moreover, we revealed a close evolutionary relationship between the isolates from different sources. B. cereus isolates from different generations worldwide showed coherence in potential pathogenicity, although with high genomic heterogeneity. The BCI-BCII-vanZF-fosB profiles and virulence and biofilm genes were detected at high rates, and we emphasized that B. cereus would pose a serious challenge to global public health and clinical treatment.

Application of a rapid and sensitive RPA-CRISPR/Cas12a assay for naked-eye detection of Haemophilus parasuis.

BACKGROUND: Haemophilus parasuis (H. parasuis) is a gram-negative bacterial pathogen that causes severe infections in swine, resulting in substantial economic losses. Currently, the majority of H. parasuis detection methods are impractical for on-site application due to their reliance on large instruments or complex procedures. Thus, there is an urgent need to develop a rapid, visually detectable, and highly sensitive detection method, especially under resource-limited environments and field conditions. RESULTS: In this study, we established a naked eye assay for highly sensitive detection by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Positive samples exhibited a clear red color visible to the naked eye, while negative samples appeared blue. We achieved a remarkable sensitivity, detecting H. parasuis down to a single copy, with no cross-reactivity with other bacteria. In a mouse model, our assay detected H. parasuis infection nearly 8 h earlier than traditional PCR. Compared to qPCR, our detection results were 100 % accurate. To enhance point-of-care applicability and mitigate the risk of aerosol contamination from uncapping, we consolidated RPA and CRISPR/Cas12a cleavage into a single-tube reaction system. This integrated approach was validated with 20 clinical lung samples, yielding results consistent with those obtained from qPCR. The entire procedure, from DNA extraction to detection, was completed in 35 min. SIGNIFICANCE: We present an RPA-CRISPR/Cas12a assay suitable for the early and resource-efficient diagnosis of H. parasuis infections. Its simplicity and visual detection are advantageous for field diagnostics, representing a substantial develpoment in the diagnosis of H. parasuis.

Prevalence and genomic characterization of the Bacillus cereus group strains contamination in food products in Southern China.

The Bacillus cereus group, as one of the important opportunistic foodborne pathogens, is considered a risk to public health due to foodborne diseases and an important cause of economic losses to food industries. This study aimed to gain essential information on the prevalence, phenotype, and genotype of B. cereus group strains isolated from various food products in China. A total of 890 strains of B. cereus group bacteria from 1181 food samples from 2020 to 2023 were identified using the standardized detection method. These strains were found to be prevalent in various food types, with the highest contamination rates observed in cereal flour (55.8 %) and wheat/rice noodles (45.7 %). The tested strains exhibited high resistance rates against penicillin (98.5 %) and ampicillin (98.9 %). Strains isolated from cereal flour had the highest rate of meropenem resistance (7.8 %), while strains from sausages were most resistant to vancomycin (16.8 %). A total of 234 out of the 891 B. cereus group strains were randomly selected for WGS analysis, 18.4 % of which displayed multidrug resistance. The species identification by WGS analysis revealed the presence of 10 distinct species within the B. cereus group, with B. cereus species being the most prevalent. The highest level of species diversity was observed in sausages. Notably, B. anthracis strains lacking the anthrax toxin genes were detected in flour-based food products and sausages. A total of 20 antibiotic resistance genes have been identified, with ß-lactam resistance genes (bla1, bla2, BcI, BcII, and blaTEM-116) being the most common. The B. tropicus strains exhibit the highest average number of virulence genes (23.4). The diarrheal virulence genes nheABC, hblACD, and cytK were found in numerous strains. Only 4 of the 234 (1.7 %) sequenced strains contain the ces gene cluster linked to emetic symptoms. These data offer valuable insights for public health policymakers on addressing foodborne B. cereus group infections and ensuring food safety.

Core and variable antimicrobial resistance genes in the gut microbiomes of Chinese and European pigs.

Monitoring the prevalence of antimicrobial resistance genes (ARGs) is vital for addressing the global crisis of antibiotic-resistant bacterial infections. Despite its importance, the characterization of ARGs and microbiome structures, as well as the identification of indicators for routine ARG monitoring in pig farms, are still lacking, particularly concerning variations in antimicrobial exposure in different countries or regions. Here, metagenomics and random forest machine learning were used to elucidate the ARG profiles, microbiome structures, and ARG contamination indicators in pig manure under different antimicrobial pressures between China and Europe. Results showed that Chinese pigs exposed to high-level antimicrobials exhibited higher total and plasmid-mediated ARG abundances compared to those in European pigs ( P<0.05). ANT(6)-Ib, APH(3')-IIIa, and tet(40) were identified as shared core ARGs between the two pig populations. Furthermore, the core ARGs identified in pig populations were correlated with those found in human populations within the same geographical regions. Lactobacillus and Prevotella were identified as the dominant genera in the core microbiomes of Chinese and European pigs, respectively. Forty ARG markers and 43 biomarkers were able to differentiate between the Chinese and European pig manure samples with accuracies of 100% and 98.7%, respectively. Indicators for assessing ARG contamination in Chinese and European pigs also achieved high accuracy ( r=0.72-0.88). Escherichia flexneri in both Chinese and European pig populations carried between 21 and 37 ARGs. The results of this study emphasize the importance of global collaboration in reducing antimicrobial resistance risk and provide validated indicators for evaluating the risk of ARG contamination in pig farms.

Application of CRISPR/Cas12a in the rapid detection of pathogens.

The combination of clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas protein is an effective gene-editing instrument. Among them, the CRISPR-Cas12a system forms a DNA-cleavage-capable complex with crRNA and exerts its trans-cleavage activity by recognising the PAM site on the target pathogen's gene. After amplifying the pathogenic gene, display materials such as fluorescent probes are added to the detection system, along with the advantages of rapid detection and high sensitivity of the CRISPR system, so that pathogenic bacteria can be diagnosed with greater speed and precision. This article reviews the mechanism of CRISPR-Cas12a in rapid detection, as well as its progress in the rapid detection of pathogenic bacteria in conjunction with various molecular biology techniques, in order to provide a foundation for the future development of a more effective detection platform.

Tigecycline Resistance-Associated Mutations in the MepA Efflux Pump in Staphylococcus aureus.

Tigecycline is an important antibacterial drug for treating infection by clinical multidrug-resistant bacteria, and tigecycline-resistant Staphylococcus aureus (TRSA) has been increasingly reported in recent years. Notably, only rpsJ and mepA are associated with the tigecycline resistance of S. aureus. The mepA gene encodes MepA efflux pumps, and the overexpression of mepA has been confirmed to be directly related to tigecycline resistance. Although the mutations of MepA widely occur, the associations between TRSA and mutations of MepA are still unclear. In this study, we explored mutations in the mepA genes from various sources. Then, tigecycline resistance-associated mutations T29I, E287G, and T29I+E287G in MepA were identified, and their effects were evaluated through mutant deletion and complementation, tigecycline accumulation assay, and molecular docking experiments. Results showed that the MICs of tigecycline, gentamicin, and amikacin increased in special complementary transformants and recovered after the addition of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The tigecycline accumulation assay of the mepA-deleted mutant strain and its complementary transformants showed that T29I, E287G, and T29I+E287G mutations promoted tigecycline efflux, and molecular docking showed that mutations T29I, E287G, and T29I+E287G decreased the binding energy and contributed to ligand binding. Moreover, we inferred the evolutionary trajectory of S. aureus under the selective pressure of tigecycline in vitro. Overall, our study indicated that mutations in MepA play important roles in tigecycline resistance in S. aureus. IMPORTANCE Previous analysis has shown that overexpression of MepA is an exact mechanism involved in tigecycline resistance apart from the rpsJ mutation and is usually dependent on the mutant mepR. However, no research has evaluated the effects of diverse mutations discovered in TRSA in MepA. This study demonstrates that the mutations in MepA confer resistance to tigecycline without overexpression and provides genotypic references for identifying TRSA. Although tigecycline resistance-associated mutations in MepA identified in this study have not been observed in clinical isolates, the mechanism should be explored given that S. aureus strains are prevalent in the environment. Measures should be implemented to contain TRSA within the time window before tigecycline resistance-associated mutations in MepA are prevalent.

Naked-eye on-site detection platform for Pasteurella multocida based on the CRISPR-Cas12a system coupled with recombinase polymerase amplification.

Pasteurella multocida (P. multocida) is an important pathogenic bacterium that poses a serious threat to the development of the livestock economy and human health. Currently, the existing methods for P. multocida detection are time-consuming and require complex professional operations, limiting the application of field detection. In the study, we presented a single-pot naked-eye CRISPR-Cas12a platform (Cas12a-NEye) for the detection of P. multocida. The round tube cover allowed more Cas12a detection solution to be temporarily stored than the flat cap, enabling single-pot assays and avoiding aerosol contamination. The positive samples generated obvious red using naked eye using no excitation light and the negative samples generated blue. The limit of detection (LOD) was a single copy, without cross-reactivity with other closely related bacteria. Furthermore, we validated this platform using 16 P. multocida clinical lung samples and obtained consistent results with the real-time quantitative polymerase chain reaction (qPCR) method. The entire experimental process included rapid DNA extraction (<1 h) and Cas12a-NEye assay (25 min), which was accomplished within 1.5 h. Thus, this "sample-to-answer" platform has significant potential for P. multocida detection.