Medicarpin suppresses lung cancer cell growth in vitro and in vivo by inducing cell apoptosis.

Lung cancer (LC) is the leading cause of cancer deaths worldwide. Surgery, chemoradiotherapy, targeted therapy, and immunotherapy are considered dominant treatment strategies for LC in the clinic. However, drug resistance and meta-stasis are two major challenges in cancer therapies. Medicarpin (MED) is an isoflavone compound isolated from alfalfa, which is usually used in traditional medicine. This study was de sig ned to evaluate the anti-LC effect and reveal the underlying mechanisms of MED in vivo and in vitro. We found that MED could significantly inhibit proliferation, induce apoptosis, and cell cycle arrest of A549 and H157 cell lines. Basically, MED induced cell apoptosis of LC cells by upregu lating the expression of pro-apoptotic proteins BAX and Bak1, leading to the cleavage of caspase-3 (Casp3). Moreover, MED inhibited the proliferation of LC cells via downregulating the expression of proliferative protein Bid. Overall, MED inhibited LC cell growth in vitro and in vivo via suppressing cell proliferation and inducing cell apoptosis, suggesting the therapeutic potential of MED in treating LC.

A Trade-Off for Maintenance of Multidrug-Resistant IncHI2 Plasmids in Salmonella enterica Serovar Typhimurium through Adaptive Evolution.

Understanding the fitness costs associated with plasmid carriage is a key to better understanding the mechanisms of plasmid maintenance in bacteria. In the current work, we performed multiple serial passages (63 days, 627.8 generations) to identify the compensatory mechanisms that Salmonella enterica serovar Typhimurium ATCC 14028 utilized to maintain the multidrug-resistant (MDR) IncHI2 plasmid pJXP9 in the presence and absence of antibiotic selection. The plasmid pJXP9 was maintained for hundreds of generations even without drug exposure. Endpoint evolved (the endpoint of evolution) S. Typhimurium bearing evolved plasmids displayed decreased growth lag times and a competitive advantage over ancestral pJXP9 plasmid-carrying ATCC 14028 strains. Genomic and transcriptomic analyses revealed that the fitness costs of carrying pJXP9 were derived from both specific plasmid genes and particularly the MDR regions and conjugation transfer region I and conflicts resulting from chromosome-plasmid gene interactions. Correspondingly, plasmid deletions of these regions could compensate for the fitness cost that was due to the plasmid carriage. The deletion extent and range of large fragments on the evolved plasmids, as well as the trajectory of deletion mutation, were related to the antibiotic treatment conditions. Furthermore, it is also adaptive evolution that chromosomal gene mutations and altered mRNA expression correlated with changed physiological functions of the bacterium, such as decreased flagellar motility, increased oxidative stress, and fumaric acid synthesis but increased Cu resistance in a given niche. Our findings indicated that plasmid maintenance evolves via a plasmid-bacterium adaptative evolutionary process that is a trade-off between vertical and horizontal transmission costs along with associated alterations in host bacterial physiology. IMPORTANCE The current idea that compensatory evolution processes can account for the "plasmid paradox" phenomenon associated with the maintenance of large costly plasmids in host bacteria has attracted much attention. Although many compensatory mutations have been discovered through various plasmid-host bacterial evolution experiments, the basis of the compensatory mechanisms and the nature of the bacteria themselves to address the fitness costs remain unclear. In addition, the genetic backgrounds of plasmids and strains involved in previous research were limited and clinical drug resistance such as the poorly understood compensatory evolution among clinically dominant multidrug-resistant plasmids or clones was rarely considered. The IncHI2 plasmid is widely distributed in Salmonella Typhimurium and plays an important role in the emergence and rapid spread of its multidrug resistance. In this study, the predominant multidrug-resistant IncHI2 plasmid pJXP9 and the standard Salmonella Typhimurium ATCC 14028 bacteria were used for evolution experiments under laboratory conditions. Our findings indicated that plasmid maintenance through experimental evolution of plasmid-host bacteria is a trade-off between increasing plasmid vertical transmission and impairing its horizontal transmission and bacterial physiological phenotypes, in which compensatory mutations and altered chromosomal expression profiles collectively contribute to alleviating plasmid-borne fitness cost. These results provided potential insights into understanding the relationship of coexistence between plasmids encoding antibiotic resistance and their bacterial hosts and provided a clue to the adaptive forces that shaped the evolution of these plasmids within bacteria and to predicting the evolution trajectory of antibiotic resistance.

Plasmid and chromosomal copies of blaCMY-2 mediate resistance to third-generation cephalosporins in Escherichia coli from food animals in China.

The use of antimicrobials in food animals is the major determinant for the propagation of resistant bacteria in the animal reservoir. The objective of this study was to investigate the presence and distribution of third-generation cephalosporin (3GC) -resistant and plasmid-mediated AmpC (pAmpC)-producing Escherichia coli isolated from food animals in Southern China. In total, 744 3GC-resistant and 40 blaCMY-2-positive E. coli strains were recovered from 1656 food animal fecal samples across five rearing regions. The blaCMY-2 genes were located on IncC, IncFIB or IncI1 type plasmids in 12 E. coli isolates. In the other 22 isolates, S1-PFGE and hybridization analyses revealed that the blaCMY-2 gene was chromosomally located and demonstrated a high prevalence of the chromosomally encoded blaCMY-2 gene in E. coli. Plasmid stability and growth curve experiments demonstrated that IncI1, IncC and IncFIB plasmids can exist stably in the host bacteria and with a low growth burden and may be the reason these plasmids can be widely disseminated in breeding environments. Whole genome sequencing indicated that ISEcp1 and IS1294 played important roles in blaCMY-2 transfer to both plasmids and the chromosome. Our study confirmed that blaCMY-2 mediated resistance of food animal-derived E. coli to 3GC and highlights the urgent need for appropriate monitoring programmes.

Formation, Transmission, and Dynamic Evolution of a Multidrug-Resistant Chromosomally Integrated Plasmid in Salmonella Spp.

IncHI2 plasmids, possessing high flexibility and genetic plasticity, play a vital role in the acquisition and transmission of resistance determinants. Polymorphic mobile genetic elements (MGEs) generated by a chromosomally integrated IncHI2 plasmid in an individual Salmonella isolate have not yet been detected, and the mechanisms of the formation, excision, and dynamic evolution of a multidrug-resistant chromosomally integrated plasmid (MRCP) have remained obscure. Herein, we identified a 260-kb bla CTX-M-55-qnrS1-bearing IncHI2 plasmid within a Salmonella Muenster strain. Plenty of heterogeneous MGEs (new Escherichia coli chromosomally integrated plasmid or circular plasmids with different profiles) were yielded when this MRCP was conjugated into E. coli J53 with a transfer frequency of 10-4-10-5 transconjugants per donor. A bioinformatic analysis indicated that replicative transposition and homologous recombination of IS26 elements were particularly active, and the truncated Tn1721 also played a vital role in the formation of MRCP offspring. More importantly, when released from the chromosome, MRCP could capture and co-transfer adjacent chromosomal segments to form larger plasmid progeny than itself. Stability and growth kinetics assays showed that the biological characteristics of MRCP progeny were differentiated. This study provides an insight into a flexible existence of MRCP. The conversion between vertical and horizontal transmission endowed MRCP with genetic stability as a chromosomal coding structure and transferability as extra-chromosomal elements. This alternation may accelerate the acquisition and persistence of antibiotic resistance of clinical pathogens and enhance their ability to respond to adverse environments, which poses a great challenge to the traditional antibiotic treatment.

IS26 Is Responsible for the Evolution and Transmission of blaNDM-Harboring Plasmids in Escherichia coli of Poultry Origin in China.

Carbapenem-resistant Enterobacteriaceae are some of the most important pathogens responsible for nosocomial infections, which can be challenging to treat. The blaNDM carbapenemase genes, which are expressed by New Delhi metallo-ß-lactamase (NDM)-producing Escherichia coli isolates, have been found in humans, environmental samples, and multiple other sources worldwide. Importantly, these genes have also been found in farm animals, which are considered an NDM reservoir and an important source of human infections. However, the dynamic evolution of blaNDM genetic contexts and blaNDM-harboring plasmids has not been directly observed, making it difficult to assess the extent of horizontal dissemination of the blaNDM gene. In this study, we detected NDM-1 (n = 1), NDM-5 (n = 24), and NDM-9 (n = 8) variants expressed by E. coli strains isolated from poultry in China from 2016 to 2017. By analyzing the immediate genetic environment of the blaNDM genes, we found that IS26 was associated with multiple types of blaNDM multidrug resistance regions, and we identified various IS26-derived circular intermediates. Importantly, in E. coli strain GD33, we propose that IncHI2 and IncI1 plasmids can fuse when IS26 is present. Our analysis of the IS26 elements flanking blaNDM allowed us to propose an important role for IS26 elements in the evolution of multidrug-resistant regions (MRRs) and in the dissemination of blaNDM. To the best of our knowledge, this is the first description of the dynamic evolution of blaNDM genetic contexts and blaNDM-harboring plasmids. These findings could help proactively limit the transmission of these NDM-producing isolates from food animals to humans. IMPORTANCE Carbapenem resistance in members of the order Enterobacterales is a growing public health problem that is associated with high mortality in developing and industrialized countries. Moreover, in the field of veterinary medicine, the occurrence of New Delhi metallo-ß-lactamase-producing Escherichia coli isolates in animals, especially food-producing animals, has become a growing concern in recent years. The wide dissemination of blaNDM is closely related to mobile genetic elements (MGEs) and plasmids. Although previous analyses have explored the association of many different MGEs with mobilization of blaNDM, little is known about the evolution of various genetic contexts of blaNDM in E. coli. Here, we report the important role of IS26 in forming multiple types of blaNDM multidrug resistance cassettes and the dynamic recombination of plasmids bearing blaNDM. These results suggest that significant attention should be paid to monitoring the transmission and further evolution of blaNDM-harboring plasmids among E. coli strains of food animal origin.

Contribution of Different Mechanisms to Ciprofloxacin Resistance in Salmonella spp.

Development of fluoroquinolone resistance can involve several mechanisms that include chromosomal mutations in genes (gyrAB and parCE) encoding the target bacterial topoisomerase enzymes, increased expression of the AcrAB-TolC efflux system, and acquisition of transmissible quinolone-resistance genes. In this study, 176 Salmonella isolates from animals with a broad range of ciprofloxacin MICs were collected to analyze the contribution of these different mechanisms to different phenotypes. All isolates were classified according to their ciprofloxacin susceptibility pattern into five groups as follows: highly resistant (HR), resistant (R), intermediate (I), reduced susceptibility (RS), and susceptible (S). We found that the ParC T57S substitution was common in strains exhibiting lowest MICs of ciprofloxacin while increased MICs depended on the type of GyrA mutation. The ParC T57S substitution appeared to incur little cost to bacterial fitness on its own. The presence of PMQR genes represented an route for resistance development in the absence of target-site mutations. Switching of the plasmid-mediated quinolone resistance (PMQR) gene location from a plasmid to the chromosome was observed and resulted in decreased ciprofloxacin susceptibility; this also correlated with increased fitness and a stable resistance phenotype. The overexpression of AcrAB-TolC played an important role in isolates with small decreases in susceptibility and expression was upregulated by MarA more often than by RamA. This study increases our understanding of the relative importance of several resistance mechanisms in the development of fluoroquinolone resistance in Salmonella from the food chain.

A Novel Structure Harboring blaCTX-M-27 on IncF Plasmids in Escherichia coli Isolated from Swine in China.

The aim of this study was to elucidate the prevalence of blaCTX-M-27-producing Escherichia coli and transmission mechanisms of blaCTX-M-27 from swine farms in China. A total of 333 E. coli isolates were collected from two farms from 2013 to 2016. Thirty-two CTX-M-27-positive E. coli were obtained, and all were multidrug-resistant. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) profiles indicated a wide range of strain types that carried blaCTX-M-27, and the sequence type ST10 predominated. Conjugation, replicon typing, S1-PFGE and hybridization experiments confirmed that 28 out of 32 CTX-M-27 positive isolates carried blaCTX-M-27 genes on plasmids F18:A-:B10 (16) and F24:A-:B1 (12).The blaCTX-M-27 genes for 24 isolates were transmitted by plasmids with sizes ranging from 40 to 155 kb. A comparative analysis with blaCTX-M-27-plasmids indicated that the tra-trb region of F24:A-:B1 plasmids was destroyed by insertion of a complex region (eight isolates) and a novel structure containing blaCTX-M-27 in the F18:A-:B10 plasmids (12 isolates). The novel structure increased the stability of the blaCTX-M-27 gene in E. coli. This study indicated that the predominant vehicle for blaCTX-M-27 transmission has diversified over time and that control strategies to limit blaCTX-M-27 transmission in farm animals are necessary.

Coordinated Expression of acrAB-tolC and Eight Other Functional Efflux Pumps Through Activating ramA and marA in Salmonella enterica serovar Typhimurium.

The aim of this study was to determine the expression of eight other functional transporter genes upon acrAB inactivation and also the expression of acrAB when the function of eight other transporters are impaired in Salmonella enterica. We used single- or multigene deletion mutants (i.e., ΔacrA, ΔacrB, ΔtolC, ΔacrAB, ΔacrEF, ΔacrD, ΔmdsABC, ΔmdtABC, ΔemrAB, ΔmacAB, ΔmdfA, ΔmdtK, ΔacrABramA, ΔacrABmarA, and ΔacrABsoxS) and real time (RT)-PCR to quantify the expression of different pump and regulator genes; infection ability was characterized by adhesion and invasion assays. The expression of acrAB operon was increased upon acrB inactivation. Single deletion of acrA or tolC also increased expression of acrB. The deletion of acrAB increased expression of eight other functional efflux pumps genes and vice versa, in which increased expression of ramA and marA was also detected. Mutants containing single deletions of functional pump genes were attenuated in cells. In conclusion, there is a feedback mechanism that coordinates regulation of AcrAB-TolC and eight other functional efflux pumps through the global transcriptional regulators ramA and marA in S. enterica serovar Typhimurium.

Upregulation of AcrEF in Quinolone Resistance Development in Escherichia coli When AcrAB-TolC Function Is Impaired.

We studied mechanisms of drug resistance development in Escherichia coli strains lacking efflux pump components. E. coli K12 deletion mutants were subjected to increasing concentrations of ciprofloxacin (CIP) to determine the frequency of target gene mutations. We generated a series of mutants that were selected based on their minimum inhibitory concentrations (MICs) to CIP, as well as their corresponding point mutations in target genes. The mutants displayed a number of target modifications and, in particular, gyrA mutations altering codons Ser83Leu, Asp87Gly, and Asp87His as well as a change in parC at 78 (substitution of Gly for Asp). All these mutations were related to drug resistance. When exposed to CIP, mutants lacking efflux pump genes acrA and acrB demonstrated a low level of resistance that was because of point mutations in the target genes. High-level resistance was achieved with a 100- to 500-fold increase in expression of efflux pump genes acrE and acrF that compensated for the loss of AcrA and AcrB, and thus resulted in an obvious increase of CIP MIC. We demonstrate that an intact AcrAB-TolC efflux pump is crucial to the development of bacterial resistance. Its activity is complemented by expression of the alternative AcrEF efflux pump.

Resistance mechanisms and fitness of Salmonella Typhimurium and Salmonella Enteritidis mutants evolved under selection with ciprofloxacin in vitro.

The aim of this study was to investigate the difference in resistance mechanisms and fitness of Salmonella Typhimurium (ST) and Salmonella Enteritidis (SE) mutants selected during the evolution of resistance under exposure to increasing ciprofloxacin concentrations in vitro. Mutations in quinolone target genes were screened by PCR. Phenotypic characterization included susceptibility testing by the broth dilution method, investigation of efflux activity and growth rate, and determination of the invasion of human intestinal epithelium cells in vitro. The two Salmonella serotypes exhibited differences in target gene mutations and efflux pump gene expression during the development of resistance. In the parental strains, ST had a competitive advantage over SE. During the development of resistance, initially, the SE strain was more competitive. However, once ciprofloxacin resistance was acquired, ST once again became the more competitive strain. In the absence of bile salts or at 0.1% bile, the growth rate of SE was initially greater than that of ST, but once ciprofloxacin resistance was acquired, ST had higher growth rates. ST strains showed decreased invasion of epithelial cells in 0.1% bile. These data indicate that ciprofloxacin-resistant ST strains are more competitive than ciprofloxacin-resistant SE strains.