Inactivation of MarR gene homologs increases susceptibility to antimicrobials in Bacteroides fragilis

ABSTRACT Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.

The prevalence of aminoglycoside-modifying enzyme and virulence genes among enterococci with high-level aminoglycoside resistance in Inner Mongolia, China

ABSTRACT This study highlights the prevalence of aminoglycoside-modifying enzyme genes and virulence determinants among clinical enterococci with high-level aminoglycoside resistance in Inner Mongolia, China. Screening for high-level aminoglycoside resistance against 117 enterococcal clinical isolates was performed using the agar-screening method. Out of the 117 enterococcal isolates, 46 were selected for further detection and determination of the distribution of aminoglycoside-modifying enzyme-encoding genes and virulence determinants using polymerase chain reaction -based methods. Enterococcus faecium and Enterococcus faecalis were identified as the species of greatest clinical importance. The aac(6')-Ie-aph(2")-Ia and ant(6')-Ia genes were found to be the most common aminoglycoside-modifying enzyme genes among high-level gentamicin resistance and high-level streptomycin resistance isolates, respectively. Moreover, gelE was the most common virulence gene among high-level aminoglycoside resistance isolates. Compared to Enterococcus faecium, Enterococcus faecalis harbored multiple virulence determinants. The results further indicated no correlation between aminoglycoside-modifying enzyme gene profiles and the distribution of virulence genes among the enterococcal isolates with high-level gentamicin resistance or high-level streptomycin resistance evaluated in our study.

Oleanolic acid and ursolic acid inhibit peptidoglycan biosynthesis in Streptococcus mutans UA159

In this study, we revealed that OA and UA significantly inhibited the expression of most genes related to peptidoglycan biosynthesis in S. mutans UA159. To the best of our knowledge, this is the first report to introduce the antimicrobial mechanism of OA and UA against S. mutans.

Genome-wide transcription analyses in Mycobacterium tuberculosis treated with lupulone

Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, still causes higher mortality than any other bacterial pathogen until now. With the emergence and spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR-TB) strains, it becomes more important to search for alternative targets to develop new antimycobacterial drugs. Lupulone is a compound extracted from Hops (Hurnulus lupulus), which exhibits a good antimicrobial activity against M. tuberculosis with minimal inhibitory concentration (MIC) value of 10 µg/mL, but the response mechanisms of lupulone against M. tuberculosis are still poorly understood. In this study, we used a commercial oligonucleotide microarray to determine the overall transcriptional response of M. tuberculosis H37Rv triggered by exposure to MIC of lupulone. A total of 540 genes were found to be differentially regulated by lupulone. Of these, 254 genes were upregulated, and 286 genes were downregulated. A number of important genes were significantly regulated which are involved in various pathways, such as surface-exposed lipids, cytochrome P450 enzymes, PE/PPE multigene families, ABC transporters, and protein synthesis. Real-time quantitative RT-PCR was performed for choosed genes to verified the microarray results. To our knowledge, this genome-wide transcriptomics approach has produced the first insights into the response of M. tuberculosis to a lupulone challenge.

Methylobacterium-plant interaction genes regulated by plant exudate and quorum sensing molecules

Bacteria from the genus Methylobacterium interact symbiotically (endophytically and epiphytically) with different plant species. These interactions can promote plant growth or induce systemic resistance, increasing plant fitness. The plant colonization is guided by molecular communication between bacteria-bacteria and bacteria-plants, where the bacteria recognize specific exuded compounds by other bacteria (e.g. homoserine molecules) and/or by the plant roots (e.g. flavonoids, ethanol and methanol), respectively. In this context, the aim of this study was to evaluate the effect of quorum sensing molecules (N-acyl-homoserine lactones) and plant exudates (including ethanol) in the expression of a series of bacterial genes involved in Methylobacterium-plant interaction. The selected genes are related to bacterial metabolism (mxaF), adaptation to stressful environment (crtI, phoU and sss), to interactions with plant metabolism compounds (acdS) and pathogenicity (patatin and phoU). Under in vitro conditions, our results showed the differential expression of some important genes related to metabolism, stress and pathogenesis, thereby AHL molecules up-regulate all tested genes, except phoU, while plant exudates induce only mxaF gene expression. In the presence of plant exudates there is a lower bacterial density (due the endophytic and epiphytic colonization), which produce less AHL, leading to down regulation of genes when compared to the control. Therefore, bacterial density, more than plant exudate, influences the expression of genes related to plant-bacteria interaction.

Streptomycin induced protein expression analysis in Mycobacterium tuberculosis by two-dimensional gel electrophoresis & mass spectrometry.

Background & objectives: The resistance of Mycobacterium tuberculosis to streptomycin, a core drug for treatment of category II tuberculosis (TB) has posed a major challenge to the health providers as well as research workers worldwide and has severely compromised the therapeutic options. A significant proportion of streptomycin resistant M. tuberculosis isolates failed to show mutations in conventional targets like rpsL and rrs. Although efflux, permeability, etc. are also known to contribute, yet a substantial proportion of isolates remains resistant suggesting involvement of other unknown mechanism. A resistant isolate may show altered gene as well as protein expression under drug induced conditions and a whole cell proteome analysis under induced conditions might help in further understanding the mechanisms of drug resistance. The present study was therefore designed with the objective to identify proteins related to streptomycin resistance in M. tuberculosis isolate grown in presence and absence of streptomycin (SM). Methods: A clinical isolate of M. tuberculosis from Mycobacterial Repository Centre at the Institute (NJIL & OMD), Agra was grown in Sauton’s medium for 36 h with/without subinhibitory concentration of the drug (2 μg/ml) and the cell lysate of isolates was prepared by sonication and centrifugation. Two-dimensional (2D) gel electrophoresis was employed to study the protein profile. The selected proteins were finally identified by MALDI-TOF mass spectrometry. Results: Our study revealed eight inducible proteins (DnaK, fabG4, DNA-binding, hypothetical, two 14 kDa antigen and two 10 kDa chaperonin) that were upregulated in the presence of drug. Interpretation & conclusion: This preliminary study has thrown light on whether or not and how the resistant isolate responds to streptomycin at its non-toxic but sub-inhibitory concentration. An in-depth study of the upregulated proteins will give an insight into probable sites of drug action other than established primary sites.