Arunachalam Muthaiyan

Transcriptional Profiling Reveals that Daptomycin Induces the Staphylococcus aureus Cell Wall Stress Stimulon and Genes Responsive to Membrane Depolarization

ABSTRACT Daptomycin is a lipopeptide antibiotic that has recently been approved for treatment of gram-positive bacterial infections. The mode of action of daptomycin is not yet entirely clear. To further understand the mechanism transcriptomic analysis of changes in gene expression in daptomycin-treated Staphylococcus aureus was carried out. The expression profile indicated that cell wall stress stimulon member genes (B. J. Wilkinson, A. Muthaiyan, and R. K. Jayaswal, Curr. Med. Chem. Anti-Infect. Agents 4:259-276, 2005) were significantly induced by daptomycin and by the cell wall-active antibiotics vancomycin and oxacillin. Comparison of the daptomycin response of a two-component cell wall stress stimulon regulator VraSR mutant, S. aureus KVR, to its parent N315 showed diminished expression of the cell wall stress stimulon in the mutant. Daptomycin has been proposed to cause membrane depolarization, and the transcriptional responses to carbonyl cyanide m-chlorophenylhydrazone (CCCP) and nisin were determined. Transcriptional profiles of the responses to these antimicrobial agents showed significantly different patterns compared to those of the cell wall-active antibiotics, including little or no induction of the cell wall stress stimulon. However, there were a significant number of genes induced by both CCCP and daptomycin that were not induced by oxacillin or vancomycin, so the daptomycin transcriptome probably reflected a membrane depolarizing activity of this antimicrobial also. The results indicate that inhibition of peptidoglycan biosynthesis, either directly or indirectly, and membrane depolarization are parts of the mode of action of daptomycin.

Effect of prebiotic carbohydrates on the growth and tolerance of Lactobacillus

Resistance to gastrointestinal conditions is a requirement for bacteria to be considered probiotics. In this work, we tested the resistance of six different Lactobacillus strains and the effect of carbon source to four different gastrointestinal conditions: presence of α-amylase, pancreatin, bile extract and low pH. Novel galactooligosaccharides synthesized from lactulose (GOS-Lu) as well as commercial galactooligosaccharides synthesized from lactose (GOS-La) and lactulose were used as carbon sources and compared with glucose. In general, all strains grew in all carbon sources, although after 24 h of fermentation the population of all Lactobacillus strains was higher for both types of GOS than for glucose and lactulose. No differences were found among GOS-Lu and GOS-La. α-amylase and pancreatin resistance was retained at all times for all strains. However, a dependence on carbon source and Lactobacillus strain was observed for bile extract and low pH resistance. High hydrophobicity was found for all strains with GOS-Lu when compared with other carbon sources. However, concentrations of lactic and acetic acids were higher in glucose and lactulose than GOS-Lu and GOS-La. These results show that the resistance to gastrointestinal conditions and hydrophobicity is directly related with the carbon source and Lactobacillus strains. In this sense, the use of prebiotics as GOS and lactulose could be an excellent alternative to monosaccharides to support growth of probiotic Lactobacillus strains and improve their survival through the gastrointestinal tract.

A Review of the Ecology, Genomics, and Stress Response of Listeria innocua and Listeria monocytogenes

Listeria monocytogenes is a Gram-positive foodborne pathogen responsible for a severe disease occurring in immuno-compromised populations. Foodborne illness caused by L. monocytogenes is a serious public health concern because of the high associated mortality. Study of the closely related, but nonpathogenic Listeria innocua has accounted for a better understanding of the behavior of L. monocytogenes in environments beyond the laboratory. Traditionally, the ecological co-habitation, genomic synteny, and physiological similarity of the two species have supported use of L. innocua for predicting the behavior of L. monocytogenes in farm and food processing environments. However, a careful review of the current literature indicates that in a given situation it may not be prudent to use L. innocua as a surrogate for L. monocytogenes without prior confirmation of their similar phenotypes, as an increasing number of studies have arisen demonstrating differences in L. monocytogenes and L. innocua stress response, and furthermore, there are differences among the L. monocytogenes subgroups. Future research should take into consideration that multiple surrogates might be required to accurately model even a single condition depending on the L. monocytogenes subgroup of interest.

Cell Wall Composition and Decreased Autolytic Activity and Lysostaphin Susceptibility of Glycopeptide-Intermediate Staphylococcus aureus

ABSTRACT The cell wall composition and autolytic properties of passage-selected glycopeptide-intermediate Staphylococcus aureus (GISA) isolates and their parent strains were studied in order to investigate the mechanism of decreased vancomycin susceptibility. GISA had relatively modest changes in peptidoglycan composition involving peptidoglycan interpeptide bridges and somewhat decreased cross-linking compared to that of parent strains. The cell wall phosphorus content of GISA strains was lower than that of susceptible parent strains, indicating somewhat lower wall teichoic acid levels in the GISA strains. Similar to whole cells, isolated crude cell walls retaining autolytic activity of GISA had drastically reduced autolytic activity compared to that of parent strains, and this arose early in the development of the GISA phenotype. This was due to an alteration in the autolytic enzymes of GISA as revealed by normal susceptibility of GISA-purified cell walls to parental strain autolysin extract and lower activity and altered peptidoglycan hydrolase activity profiles in GISA autolysin extracts compared to those of parent strains. Northern blot analysis indicated that expression of atl, the major autolysin gene, was significantly downregulated in a GISA strain compared to that of its parent strain. In contrast to whole cells, which showed decreased lysostaphin susceptibility, purified cell walls of GISA showed increased susceptibility to lysostaphin. We suggest that in our GISA strains, decreased autolytic activity is involved in the tolerance of vancomycin and the activities of endogenous autolysins are important in conferring sensitivity to lysostaphin on whole cells.

Transcriptomic response of Listeria monocytogenes to iron limitation and Fur mutation.

ABSTRACT Iron is required by almost all bacteria, but concentrations above physiological levels are toxic. In bacteria, intracellular iron is regulated mostly by the ferric uptake regulator, Fur, or a similar functional protein. Iron limitation results in the regulation of a number of genes, especially those involved in iron uptake. A subset of these genes is the Fur regulon under the control of Fur. In the present study, we have identified Fur- and iron-regulated genes in Listeria monocytogenes by DNA microarray analysis using a fur mutant and its isogenic parent. To identify genes regulated exclusively in response to iron limitation, the whole-genome transcriptional responses to the iron limitation of a fur mutant and its isogenic parent were compared. Fur-regulated genes were identified by comparing the transcriptional profile of the parent with the transcriptional profile of the isogenic fur mutant. Our studies have identified genes regulated exclusively in response to iron and those that are negatively regulated by Fur. We have identified at least 14 genes that were negatively regulated directly by Fur. Under iron-limited conditions, these genes were upregulated, while the expression of fur was found to be downregulated. To further investigate the regulation of fur in response to iron, an ectopic fur promoter-lacZ transcriptional fusion strain was constructed, and its isogenic fur and perR mutant derivatives were generated in L. monocytogenes 10403S. Analysis of the iron limitation of the perR mutant indicated that the regulation of genes under the negative control of Fur was significantly inhibited. Our results indicate that Fur and PerR proteins negatively regulate fur and that under iron-limited conditions, PerR is required for the negative regulation of genes controlled by Fur.

Antimicrobial activity of commercial citrus-based natural extracts against Escherichia coli O157:H7 isolates and mutant strains.

Due to increasing concerns about the development of antimicrobial resistance amongst pathogenic bacteria, alternative strategies have been sought that do not use antibiotics to reduce pathogenic bacteria from foods and patients. A natural compound that has potent antimicrobial properties is citrus peel, which contains a variety of essential oils that inhibit the growth of or kill pathogenic bacteria. In the present study, seven citrus-based natural antimicrobials were evaluated for their ability to inhibit the growth of the pathogen Escherichia coli O157:H7. Zones of inhibition of E. coli O157:H7 by the citrus-derived fraction (10 microL/6 mm disk) were determined by a disk-diffusion assay on Sorbitol-MacConkey agar. Inhibition zones were observed after 48 h lawn growth of E. coli O157:H7 cells at 37 degrees C. Two citrus-based fractions, orange CP VAL terpeneless FAB 968611 and Limonene 1x Dist FAB 955430, inhibited E. coli O157:H7 with inhibition zones of approx. 11-24 mm dia. The remaining other five citrus-derived extracts (orange oil FL VAL 1121 ARR 974760, Orange 5x Conc VAL 4121 ARR 968374, orange terpenes ESS 1120 ARR 986259, orange terpenes CP 1100 ARR 986255, and orange terpenes OEO HP 1100 ARR 986257) were noninhibitory to E. coli O157:H7, yielding no clear inhibition zones. These studies show that citrus-derived natural compounds differ in their inhibitory activity against E. coli O157:H7 and some have potential applications as inhibitory agents against E. coli O157:H7 in various pathogen reduction strategies.

Antimicrobial effect and mode of action of terpeneless cold‐pressed Valencia orange essential oil on methicillin‐resistant Staphylococcus aureus

Aims:  The objectives of this study were to evaluate the antistaphylococcal effect and elucidate the mechanism of action of orange essential oil against antibiotic‐resistant Staphylococcus aureus strains.

Effect of sublethal heat stress on Salmonella Typhimurium virulence

Aims:  To determine the virulence gene expression of Salmonella Typhimurium in response to sublethal heat stress and determine the adhesion and invasion pattern of heat‐stressed Salmonella in Caco‐2 intestinal epithelial cells.

Listeria monocytogenes: Antibiotic Resistance in Food Production

Listeria monocytogenes is an opportunistic human pathogen that causes listeriosis, a disease that mainly affects the immunocompromised, the elderly, infants, and pregnant women. Listeriosis has become increasingly common in the last 25 years since the first foodborne outbreak was noted. Treatment for listeriosis currently consists primarily of supportive therapy in conjunction with the use of intravenous antibiotics. Antibiotics have been commercially available for over 60 years for treatment of a myriad of clinical diseases. Bacteria resistant to antibiotics have been developing over this same period. This review seeks to elucidate the extent of antibiotic resistance in L. monocytogenes, the possible transmission mechanisms, and contributing factors to distribution of antibiotic resistance among Listeria species, and possible control strategies.

Genomic and proteomic analysis of the Alkali-Tolerance Response (AlTR) in Listeria monocytogenes 10403S

BackgroundInformation regarding the Alkali-Tolerance Response (AlTR) in Listeria monocytogenes is very limited. Treatment of alkali-adapted cells with the protein synthesis inhibitor chloramphenicol has revealed that the AlTR is at least partially protein-dependent. In order to gain a more comprehensive perspective on the physiology and regulation of the AlTR, we compared differential gene expression and protein content of cells adapted at pH 9.5 and un-adapted cells (pH 7.0) using complementary DNA (cDNA) microarray and two-dimensional (2D) gel electrophoresis, (combined with mass spectrometry) respectively.ResultsIn this study, L. monocytogenes was shown to exhibit a significant AlTR following a 1-h exposure to mild alkali (pH 9.5), which is capable of protecting cells from subsequent lethal alkali stress (pH 12.0). Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. The observed variability between results of cDNA arrays and 2D gel electrophoresis may be accounted for by posttranslational modifications. Interestingly, several alkali induced genes/proteins can provide a cross protective overlap to other types of stresses.ConclusionAlkali pH provides therefore L. monocytogenes with nonspecific multiple-stress resistance that may be vital for survival in the human gastrointestinal tract as well as within food processing systems where alkali conditions prevail. This study showed strong evidence that the AlTR in L. monocytogenes functions as to minimize excess alkalisation and energy expenditures while mobilizing available carbon sources.