The Fe-S cluster biosynthesis in Enterococcus faecium is essential for anaerobic growth and gastrointestinal colonization.

The facultative anaerobic Gram-positive bacterium Enterococcus faecium is a ubiquitous member of the human gut microbiota. However, it has gradually evolved into a pathogenic and multidrug resistant lineage that causes nosocomial infections. The establishment of high-level intestinal colonization by enterococci represents a critical step of infection. The majority of current research on Enterococcus has been conducted under aerobic conditions, while limited attention has been given to its physiological characteristics in anaerobic environments, which reflects its natural colonization niche in the gut. In this study, a high-density transposon mutant library containing 26,620 distinct insertion sites was constructed. Tn-seq analysis identified six genes that significantly contribute to growth under anaerobic conditions. Under anaerobic conditions, deletion of sufB (encoding Fe-S cluster assembly protein B) results in more extensive and significant impairments on carbohydrate metabolism compared to aerobic conditions. Consistently, the pathways involved in this utilization-restricted carbohydrates were mostly expressed at significantly lower levels in mutant compared to wild-type under anaerobic conditions. Moreover, deletion of sufB or pflA (encoding pyruvate formate lyase-activating protein A) led to failure of gastrointestinal colonization in mice. These findings contribute to our understanding of the mechanisms by which E. faecium maintains proliferation under anaerobic conditions and establishes colonization in the gut.

LsrR, the effector of AI-2 quorum sensing, is vital for the H2O2 stress response in mammary pathogenic Escherichia coli.

Mammary pathogenic Escherichia coli (MPEC) is an important causative agent of mastitis in dairy cows that results in reduced milk quality and production, and is responsible for severe economic losses in the dairy industry worldwide. Oxidative stress, as an imbalance between reactive oxygen species (ROS) and antioxidants, is a stress factor that is common in most bacterial habitats. The presence of ROS can damage cellular sites, including iron-sulfur clusters, cysteine and methionine protein residues, and DNA, and may cause bacterial cell death. Previous studies have reported that Autoinducer 2 (AI-2) can regulate E. coli antibiotic resistance and pathogenicity by mediating the intracellular receptor protein LsrR. This study explored the regulatory mechanism of LsrR on the H2O2 stress response in MPEC, showing that the transcript levels of lsrR significantly decreased under H2O2 stress conditions. The survival cell count of lsrR mutant XW10/pSTV28 was increased about 3080-fold when compared with that of the wild-type WT/pSTV28 in the presence of H2O2 and overexpression of lsrR (XW10/pUClsrR) resulted in a decrease in bacterial survival rates under these conditions. The ß-galactosidase reporter assays showed that mutation of lsrR led to a remarkable increase in expression of the promoters of ahpCF, katG and oxyR, while lsrR-overexpressing significantly reduced the expression of ahpCF and katG. The electrophoretic mobility shift assays confirmed that LsrR could directly bind to the promoter regions of ahpCF and katG. These results revealed the important role played by LsrR in the oxidative stress response of MPEC.

Mollicellins S-U, three new depsidones from Chaetomium brasiliense SD-596 with anti-MRSA activities.

Fungi are important resources for drug development, as they have a diversity of genes, that can produce novel secondary metabolites with effective bioactivities. Here, five depsidone-based analogs were isolated from the rice media of Chaetomium brasiliense SD-596. Their structures were elucidated using NMR and mass spectrometry analysis. Five compounds, including three new depsidone analogs, mollicellin S (1), mollicellin T (2), and mollicellin U (3), and two known compounds, mollicellin D (4) and mollicellin H (5), exhibited significant inhibition against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), with MIC values ranging from 6.25 to 12.5 µg ml-1. Herein, we identified the predicted plausible biosynthetic cluster of the compounds and discussed the structure-activity relationship. Finally, we found that the introduction of aldehyde and methoxyl groups provide marked improvement for the inhibition against MRSA.

Role of McbR in the regulation of antibiotic susceptibility in avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes a variety of bacterial infectious diseases known as avian colibacillosis leading to significant economic losses in the poultry industry worldwide and restricting the development of the poultry industry. The development of efflux pumps is one important bacterial antibiotic resistance mechanism. Efflux pumps are capable of extruding a wide range of antibiotics out of the cytoplasm of some bacterial species, including ß-lactams, polymyxins, tetracyclines, fluoroquinolones, aminoglycosides, novobiocin, nalidixic acid, and fosfomycin. In the present study, we constructed the mcbR mutant and the mcbR-overexpressing strain of E. coli strain APECX40 and performed antimicrobial susceptibility testing, antibacterial activity assays, real-time reverse transcription PCR, and electrophoretic mobility shift assays (EMSA) to investigate the molecular regulatory mechanism of McbR on the genes encoding efflux pumps. Our results showed that McbR positively regulates cell susceptibility to 12 antibiotics, including clindamycin, lincomycin, cefotaxime, cefalexin, doxycycline, tetracycline, gentamicin, kanamycin, norfloxacin, ofloxacin, erythromycin, and rifampicin by activating the transcription of acrAB, acrD, emrD, and mdtD (P < 0.01). Additionally, EMSA indicated that McbR specifically binds to the promoter regions of acrAB, acrD, acrR, emrD, and mdtD. This study suggests that, in APECX40, McbR plays an important role in the regulation of bacterial susceptibility by directly activating the transcription of efflux pumps genes.

The two-component system, BasSR, is involved in the regulation of biofilm and virulence in avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is a subgroup of extra-intestinal pathogenic E. coli (ExPEC) strains that cause avian colibacillosis, resulting in significant economic losses to the poultry industry worldwide. It has been reported that a few two-component signal transduction systems (TCS) participate in the regulation of the virulence factors of APEC infection. In this study, a basSR-deficient mutant strain was constructed from its parent strain APECX40 (WT), and high-throughput sequencing (RNA-seq) was performed to analyse the transcriptional profile of WT and its mutant strain XY1. Results showed that the deletion of basSR down-regulated the transcript levels of a series of biofilm- and virulence-related genes. Results of biofilm formation assays and bird model experiments indicated that the deletion of basSR inhibited biofilm formation in vitro and decreased bacterial virulence and colonization in vivo. In addition, electrophoretic mobility shift assays confirmed that the BasR protein could bind to the promoter regions of several biofilm- and virulence-related genes, including ais, opgC and fepA. This study suggests that the BasSR TCS might be a global regulator in the pathogenesis of APEC infection. RESEARCH HIGHLIGHTS Transcriptional profiling showed that BasSR might be a global regulator in APEC. BasSR increases APEC pathogenicity in vivo. BasSR positively regulates biofilm- and the virulence-associated genes. BasSR can bind to the promoter regions of virulence-associated genes ais, opgC and fepA.

Role of LsrR in the regulation of antibiotic sensitivity in avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is a specific group of extraintestinal pathogenic E. coli that causes a variety of extraintestinal diseases in chickens, ducks, pigeons, turkeys, and other avian species. These diseases lead to significant economic losses in the poultry industry worldwide. However, owing to excessive use of antibiotics in the treatment of infectious diseases, bacteria have developed antibiotic resistance. The development of multidrug efflux pumps is one important bacterial antibiotic resistance mechanism. A multidrug efflux pump, MdtH, which belongs to the major facilitator superfamily of transporters, confers resistance to quinolone antibiotics such as norfloxacin and enoxacin. LsrR regulates hundreds of genes that participate in myriad biological processes, including mobility, biofilm formation, and antibiotic susceptibility. However, whether LsrR regulates mdtH transcription and then affects bacterial resistance to various antibiotics in APEC has not been reported. In the present study, the lsrR mutant was constructed from its parent strain APECX40 (WT), and high-throughput sequencing was performed to analyze the transcriptional profile of the WT and mutant XY10 strains. The results showed that lsrR gene deletion upregulated the mdtH transcript level. Furthermore, we also constructed the lsrR- and mdtH-overexpressing strains and performed antimicrobial susceptibility testing, antibacterial activity assays, real-time reverse transcription PCR, and electrophoretic mobility shift assays to investigate the molecular regulatory mechanism of LsrR on the MdtH multidrug efflux pump. The lsrR mutation and the mdtH-overexpressing strain decreased cell susceptibility to norfloxacin, ofloxacin, ciprofloxacin, and tetracycline by upregulating mdtH transcript levels. In addition, the lsrR-overexpressing strain increased cell susceptibility to norfloxacin, ofloxacin, ciprofloxacin, and tetracycline by downregulating mdtH transcript levels. Electrophoretic mobility shift assays indicated that LsrR directly binds to the mdtH promoter. Therefore, this study is the first to demonstrate that LsrR inhibits mdtH transcription by directly binding to its promoter region. This action subsequently increases susceptibility to the aforementioned four antibiotics in APECX40.

Regulatory Role of the Two-Component System BasSR in the Expression of the EmrD Multidrug Efflux in Escherichia coli.

Due to excessive use of antimicrobial agents in the treatment of infectious diseases, bacteria have developed resistance to antibacterial drugs and toxic compounds. The development of multidrug efflux pumps is one of the important mechanisms of bacterial drug resistance. A multidrug efflux pump, EmrD, belonging to the major facilitator superfamily of transporters, confers resistance to many antimicrobial agents. BasSR, a typical two-component signal transduction system (TCS), regulates susceptibility to the cationic antimicrobial peptide, polymyxin B, and the anionic bile detergent, deoxycholic acid, in Escherichia coli. However, whether or not the BasSR TCS affects susceptibility or resistance to other antimicrobial agents and transcription of emrD has not been reported in E. coli. In the present study, we constructed the basSR mutants of wild-type MG1655 and clinical strain APECX40 and performed antimicrobial susceptibility testing, antibacterial activity assays, real-time reverse transcription-PCR experiments and electrophoretic mobility shift assays (EMSA) to investigate the molecular mechanism by which BasSR regulates the EmrD multidrug efflux pump. Results showed that the basSR mutation increased cell susceptibility to eight antimicrobial agents, including ciprofloxacin, norfloxacin, doxycycline, tetracycline, clindamycin, lincomycin, erythromycin, and sodium dodecyl sulfate, by downregulating the transcriptional levels of emrD. Furthermore, EMSA indicated that BasR could directly bind to the emrD promoter. Therefore, this study was the first to demonstrate that BasSR activates transcription of emrD by binding directly to its promoter region, and then decreases susceptibility to various antimicrobial agents in E. coli strains, APECX40 and MG1655.

QseBC is involved in the biofilm formation and antibiotic resistance in Escherichia coli isolated from bovine mastitis.

BACKGROUND: Mastitis is one of the most common infectious diseases in dairy cattle and causes significant financial losses in the dairy industry worldwide. Antibiotic therapy has been used as the most effective strategy for clinical mastitis treatment. However, due to the extensive use of antibacterial agents, antimicrobial resistance (AMR) is considered to be one of the reasons for low cure rates in bovine mastitis. In addition, biofilms could protect bacteria by restricting antibiotic access and shielding the bacterial pathogen from mammary gland immune defences. The functional mechanisms of quorum sensing E. coli regulators B an d C (QseBC) have been well studied in E. coli model strains; however, whether QseBC regulates antibiotic susceptibility and biofilm formation in clinical E. coli strain has not been reported. METHODS: In this study, we performed construction of the qseBC gene mutant, complementation of the qseBC mutant, antimicrobial susceptibility testing, antibacterial activity assays, biofilm formation assays, real-time reverse transcription PCR (RT-PCR) experiments and electrophoretic mobility shift assays (EMSAs) to investigate the role of qseBC in regulating biofilm formation and antibiotic susceptibility in the clinical E. coli strain ECDCM2. RESULTS: We reported that inactivation of QseBC led to a decrease in biofilm formation capacity and an increase in antibiotic susceptibility of an E. coli strain isolated from a dairy cow that suffered from mastitis. In addition, this study indicated that QseBC increased biofilm formation by upregulating the transcription of the biofilm-associated genes bcsA, csgA, fliC, motA, wcaF and fimA and decreased antibiotic susceptibility by upregulating the transcription of the efflux-pump-associated genes marA, acrA, acrB, acrD, emrD and mdtH. We also performed EMSA assays, and the results showed that QseB can directly bind to the marA promoter. CONCLUSIONS: The QseBC two-component system affects antibiotic sensitivity by regulating the transcription of efflux-pump-associated genes. Further, biofilm-formation-associated genes were also regulated by QseBC TCS in E. coli ECDCM2. Hence, this study might provide new clues to the prevention and treatment of infections caused by the clinical E. coli strains.

McbR is involved in biofilm formation and H2O2 stress response in avian pathogenic Escherichia coli X40.

Avian pathogenic Escherichia coli (APEC) causes a variety of extraintestinal diseases known as colibacillosis and is responsible for significant economic losses in the poultry industry worldwide. Biofilm formation results in increased morbidity and persistent infections, and is the main reason for the difficult treatment of colibacillosis with antimicrobial agents. It is reported that the transcriptional regulator McbR regulates biofilm formation and mucoidy by repressing the expression of the periplasmic protein YbiM, and activates the transcription of the yciGFE operon by binding to the yciG promoter in E. coli K-12. However, whether McbR regulates biofilm formation and H2O2 stress response in APEC has been not reported. The present study showed that, in the clinical isolate APECX40, the deletion of mcbR increased biofilm formation by upregulating the transcription of the biofilm-associated genes bcsA, fliC, wcaF, and fimA. In addition, the deletion of mcbR decreased H2O2 stress response by downregulating the transcript levels of the stress-associated genes yciF and yciE. The electrophoretic mobility shift assays confirmed that McbR directly binds to the promoter regions of yciG and yciF. This study may provide new clues to understanding gene regulation in APEC.

Effects of stigmata maydis on the methicillin resistant Staphylococus aureus biofilm formation.

BACKGROUND: Mastitis is an inflammatory reaction of the mammary gland tissue, which causes huge losses to dairy farms throughout the world. Staphylococcus aureus is the most frequent agent associated with this disease. Staphylococcus aureus isolates, which have the ability to form biofilms, usually lead to chronic mastitis in dairy cows. Moreover, methicillin resistance of the bacteria further complicates the treatment of this disease. Stigmata maydis (corn silk), a traditional Chinese medicine, possess many biological activities. METHODS: In this study, we performed antibacterial activity assays, biofilm formation assays and real-time reverse transcription PCR experiments to investigate the effect of stigmata maydis (corn silk) on biofilm formation and vancomycin susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from dairy cows with mastitis. RESULTS: In this study, the aqueous extracts of stigmata maydis inhibited the biofilm formation ability of MRSA strains and increased the vancomycin susceptibility of the strains under biofilm-cultured conditions. CONCLUSION: This study proves that the aqueous extracts of stigmata maydis inhibit the biofilm formation ability of MRSA strains and increase the vancomycin susceptibility of the MRSA strains under biofilm-cultured conditions.

The anti-biofilm effect of silver-nanoparticle-decorated quercetin nanoparticles on a multi-drug resistant Escherichia coli strain isolated from a dairy cow with mastitis.

BACKGROUND: Escherichia coli is an important opportunistic pathogen that could cause inflammation of the udder in dairy cows resulting in reduced milk production and changes in milk composition and quality, and even death of dairy cows. Therefore, mastitis is the main health issue which leads to major economic losses on dairy farms. Antibiotics are routinely used for the treatment of bovine mastitis. The ability to form biofilm increases the antibiotic resistance of E. coli. Nanoparticles (NPs), a nanosized, safe, and highly cost-effective antibacterial agent, are potential biomedical tools. Given their antibacterial activities, silver nanoparticles (Ag NPs) have a broad range of applications. METHODS: In this study, we performed antibacterial activity assays, biofilm formation assays, scanning electron microscopy (SEM) experiments, and real-time reverse transcription PCR (RT-PCR) experiments to investigate the antibacterial and anti-biofilm effect of quercetin, Ag NPs, and Silver-nanoparticle-decorated quercetin nanoparticles (QA NPs) in E. coli strain ECDCM1. RESULTS: In this study, QA NPs, a composite material combining Ag NPs and the plant-derived drug component quercetin, exhibited stronger antibacterial and anti-biofilm properties in a multi-drug resistant E. coli strain isolated from a dairy cow with mastitis, compared to Ag NPs and Qe. DISCUSSION: This study provides evidence that QA NPs possess high antibacterial and anti-biofilm activities. They proved to be more effective than Ag NPs and Qe against the biofilm formation of a multi-drug resistant E. coli isolated from cows with mastitis. This suggests that QA NPs might be used as a potential antimicrobial agent in the treatment of bovine mastitis caused by E. coli.

Autoinducer2 affects trimethoprim-sulfamethoxazole susceptibility in avian pathogenic Escherichia coli dependent on the folate synthesis-associate pathway.

Avian pathogenic Escherichia coli (APEC) causes airsacculitis, polyserositis, septicemia, and other mainly extraintestinal diseases in chickens, ducks, geese, pigeons, and other avian species, and is responsible for great economic losses in the avian industry. The autoinducer 2 (AI-2) quorum sensing system is widely present in many species of gram-negative and gram-positive bacteria and has been proposed to be involved in interspecies communication. In clinical APEC strains, whether or not AI-2 affects the expression of antibiotic-related genes has not been reported. In this study, we have reported that exogenous AI-2 increase the susceptibility of APEC strains to trimethoprim-sulfamethoxazole (SXT) in a folate synthesis-dependent pathway but not in the LsrR-dependent manner. Our results further explained that exogenous AI-2 can down regulate the transcription of the folate synthetase encoding genes folA and folC, and the folate synthesis-related genes luxS, metE, and metH. Gel shift assays confirmed that LsrR, the AI-2 receptor, did not bind to the promoters of folA and folC, suggesting that exogenous AI-2 might influence folate metabolism by a feedback inhibition effect but not in the LsrR-dependent pathway. This study might provide further information in the search for potential drug targets for prophylaxis of avian colibacillosis and for auxiliary antibiotics in the treatment of avian colibacillosis.

Imidazole decreases the ampicillin resistance of an Escherichia coli strain isolated from a cow with mastitis by inhibiting the function of autoinducer 2.

Extended-spectrum ß-lactamase-positive Escherichia coli is an important causative agent of mastitis in dairy cows that results in reduced milk production and quality, and is responsible for severe economic losses in the dairy industry worldwide. The quorum sensing signaling molecule autoinducer 2 (AI-2) is produced by many species of gram-negative and gram-positive bacteria, and might be a universal language for intraspecies and interspecies communication. Our previous work confirmed that exogenous AI-2 increases the antibiotic resistance of extended-spectrum ß-lactamase-positive E. coli to the ß-lactam group of antibiotics by upregulating the expression of the TEM-type ß-lactamase. In addition, this regulation relies on the function of the intracellular AI-2 receptor LsrR. In the present work, we reported that exogenous imidazole, a furan carbocyclic analog of AI-2, decreases the antibiotic resistance of a clinical E. coli strain to ß-lactam antibiotics by inhibiting the function of AI-2.

Short communication: The role of autoinducer 2 (AI-2) on antibiotic resistance regulation in an Escherichia coli strain isolated from a dairy cow with mastitis.

Extended spectrum ß-lactamase (ESBL)-positive Escherichia coli is a major etiological organism responsible for bovine mastitis. The autoinducer 2 (AI-2) quorum sensing system is widely present in many species of gram-negative and gram-positive bacteria and has been proposed to be involved in interspecies communication. In E. coli model strains, the functional mechanisms of AI-2 have been well studied; however, in clinical antibiotic-resistant E. coli strains, whether AI-2 affects the expression of antibiotic resistance genes has not been reported. In this study, we report that exogenous AI-2 increased the antibiotic resistance of a clinical E. coli strain isolated from a dairy cow with mastitis by upregulating the expression of TEM-type enzyme in an LsrR (LuxS regulated repressor)-dependent manner.