Cloning of the replication region on the bacteriocinogenic plasmid pRJ9 of Staphylococcus aureus

Abstract A 5.8-kb Cla I fragment of pRJ9, a bacteriocinogenic plasmid of Sphylococcus aureus , was cloned in the unique Cla I site of pRJ5. The recombinant plasmid obtained, pRJ23, failed to confer bacteriocin production and immunity to bacteriocin on host cells. The cloned fragment was shown to contain the complete replicon of pRJ9. Attempts to clone the 4.4-kb Cla I fragment of pRJ9 were unsuccessful, apparently due to the inactivation of the basic replicon of the cloning vector. Therefore, plasmid pRJ5 cut at its Cla I site appears to be a suitable vector for cloning replication regions of plasmids that cab replicate in S. aureus .     

Structural relationships among chloramphenicol-resistance plasmids of Staphylococcus aureus

Abstract Twelve different chloramphenicol-resistance (Cm^r) plasmids detected in Staphylococcus aureus strains isolated between 1952 and 1981 were characterized by restriction endonuclease, DNA hybridization and heteroduplex analyses. These studies revealed three families of Cm^r plasmids which were distinguished by their chloramphenicol acetyltransferase sequences; the prototype plasmids of the families were pC221, pC223 and pC194. The cat and replication ( rep ) genes of the plasmid pC221 were highly conserved in other pC221 family members and were related to their homologs in the pC223 family plasmids; however, the cat and rep genes of the pC194 family plasmids were distinct.     

Iron Acquisition and Transport in Staphylococcus aureus

Pathogenic Gram-positive bacteria encounter many obstacles in route to successful invasion and subversion of a mammalian host. As such, bacterial species have evolved clever ways to prevent the host from clearing an infection, including the production of specialized virulence systems aimed at counteracting host defenses or providing protection from host immune mechanisms. Positioned at the interface of bacteria/host interactions is the bacterial cell wall, a dynamic surface organelle that serves a multitude of functions, ranging from physiologic processes such as structural scaffold and barrier to osmotic lysis to pathogenic properties, for example the deposition of surface molecules and the secretion of cytotoxins. In order to succeed in a battle with host defenses, invading bacteria need to acquire the nutrient iron, which is sequestered within host tissues. A cell-wall based iron acquisition and import pathway was uncovered in Staphylococcus aureus. This pathway, termed the isd or iron-responsive surface determinant locus, consists of a membrane transporter, cell wall anchored heme-binding proteins, heme/haptoglobin receptors, two heme oxygenases, and sortase B, a transpeptidase that anchors substrate proteins to the cell wall. Identification of the isd pathway provides an additional function to the already bountiful roles the cell wall plays in bacterial pathogenesis and provides new avenues for therapeutics to combat the rise of antimicrobial resistance in S. aureus. This review focuses on the molecular attributes of this locus, with emphasis placed on the mechanism of iron transport and the role of such a system during infection.     

Identification and typing of food-borne Staphylococcus aureus by PCR-based techniques

The possibility of using PCR for rapid identification of food-borne Staphylococcus aureus isolates was evaluated as an alternative to the API-Staph system. A total of 158 strains, 15 S. aureus, 12 other staphylococcal species, and 131 isolates recovered from 164 food samples were studied. They were phenotypically characterized by API-Staph profiles and tested for PCR amplification with specific primers directed to thermonuclease (nuc) and enterotoxin (sea to see) genes. Disagreement between the PCR results and API-Staph identification was further assessed by the analysis of randomly amplified polymorphic DNA (RAPD) profiles obtained with three universal primers (M13, T3, and T7) and 16S rDNA sequencing. Forty out of 131 isolates (31%) tested positive for PCR enterotoxin. Of these, 14 (11%) were positive for sea, 22 (17%) for sec, one (0.8%) for sed, and three (2.2%) for sea and sec. No amplification corresponding to seb nor see was obtained. Cluster analysis based on RAPD profiles revealed that most of the sec positive food isolates grouped together in three clusters. Cluster analysis combining the three RAPD fingerprints (M 13, T3, and T7), PCR-enterotoxin genotype and API-Staph profiles, grouped the nuc PCR positive isolates together with S. aureus reference strains and the nuc PCR negative isolates with reference strains of other staphylococcal species. The only nuc PCR positive food isolate that remained unclustered was a sed positive strain identified by 16S rDNA sequence as S. simulans. The high concordance between S. aureus and nuc PCR positive strains (99%) corroborates the specificity of the primers used and the suitability of nuc PCR for rapid identification of S. aureus in routine food analysis.     

RAPD-PCR analysis of Staphylococcus aureus strains isolated from bovine and human hosts

Staphylococcus aureus is one of the most important pathogens in humans and animals. In this study eighty strains were analyzed by RAPD-PCR to assess the genetic relationship between S. aureus isolates from bovine and human hosts. Results were compared with those obtained by biotyping. Fifty-two percent of the S. aureus isolates belonged to a host specific biotype (human, bovine and poultry). Bovine and human ecovars were the most prevalent. Dendrogram obtained by RAPD results showed that all the isolates clustered into eleven groups (A-K) at a relative genetic similarity of less than 30% when analyzed with the three primers. Group A clustered 95% of the human host isolates and the remaining groups (B-K) clustered the bovine host isolates. Principal coordinate analysis also showed that the isolates could be arbitrarily divided into two groups, bovine and human, by the second coordinate. Only 9 isolates (11%) were not clustered into these groups. The genetic diversity among the S. aureus isolates from bovine hosts is relatively low compared to that of isolates from human hosts. There were no statistically significant differences among isolated from bovine and human hosts. This study shows that RAPD-PCR assayed with three primers can be successfully applied to assess the genetic relationship of S. aureus isolates from different hosts.     

Incompatibility and molecular relationships between small staphylococcal plasmids carrying the same resistance marker

Incompatibility relationships between staphylococcal plasmids carrying the same, single resistance marker were studied by means of appropriate recombinant plasmids. Naturally occurring plasmids encoding streptomycin, tetracycline, or chloramphenicol resistance, respectively, were used in this study, four of each phenotype. The plasmids responsible for tetracycline resistance proved to belong to a single incompatibility set. Similarly, the four streptomycin resistance plasmids fall in the same incompatibility set. On the other hand, plasmids encoding chloramphenicol resistance were divided in four distinct incompatibility sets, three of them being newly defined. Study of the molecular relationships between these plasmids by DNA-DNA hybridization and restriction endonuclease cleavage supported the conclusions from genetic tests that the four Tc^r and the four Sm^r plasmids are essentially identical, whereas the four Cm^r plasmids are diverse.     

Antibacterial activities of essential oils from eight Greek aromatic plants against clinical isolates of Staphylococcus aureus

Aromatic plants have been used widely to extend the shelf life of foods but at the same time research is undergoes for their properties as antibacterial agents in clinical use. Although there are promising results for the antimicrobial properties of various essential oils against environmental or food-isolated strains of Staphylococcus aureus, limited work has been done concerning these properties against clinical isolates of this pathogen. S. aureus is responsible for an increase number of nosocomial infections and at the same time exhibits increased resistance to synthetic agents.In this study, essential oils from eight aromatic plants common in Greece were isolated by hydrodistillation, analyzed by gas chromatography (GC) and GC/mass spectrometry (GC/MS) for their chemical components and tested for their antimicrobial activities against 24 clinical isolates of S. aureus. The methods used were disk diffusion and broth dilution in order to determine the Minimum Inhibitory Concentration (MIC).Our results showed that essential oils from Origanum vulgare and Origanum dictamnus were active against S. aureus when tested by disk diffusion, but exhibited increased MIC values (>256 mg/L) with the dilution method. In contrast, the reference strain NCTC 6571 showed to be extremely sensitive in most of the oils tested (MICs 0.25−32.0 mg/L) and resistant only to the essential oil from Ocimum basilicum. Therefore, there is no evidence of a potential clinical use for those essential oils and further research is needed in order to determine if they could substitute efficiently synthetic antibiotics or, perhaps be used in combination.     

Understanding the rise of the superbug: investigation of the evolution and genomic variation of Staphylococcus aureus

The bacterium Staphylococcus aureus is a common cause of human infection, and it is becoming increasingly virulent and resistant to antibiotics. Our understanding of the evolution of this species has been greatly enhanced by the recent sequencing of the genomes of seven strains of S. aureus. Comparative genomic analysis allows us to identify variation in the chromosomes and understand the mechanisms by which this versatile bacterium has accumulated diversity within its genome structure.     

The Effect of the Intra- and Extracellular Metabolites of Microorganisms Isolated from Various Ecotopes on the Catalase Activity of Staphylococcus aureus ATCC 6538 P

The cell extracts (i.e., intracellular metabolites) and culture liquids (i.e., extracellular metabolites) of microorganisms isolated from various ecotopes were found to inhibit the catalase activity of Staphylococcus aureus ATCC 6538 P, which resulted in a considerable inhibition of the growth of metabolite-treated S. aureus cells by hydrogen peroxide. The inhibitory effect of microbial metabolites on S. aureus catalase can be considered as a mechanism of intercellular interactions responsible for the formation of microbiocenoses.     

Lipids and DNA oxidation in Staphylococcus aureus as a consequence of oxidative stress generated by ciprofloxacin

Ciprofloxacin induced an increment of reactive oxygen species in sensitive strains of Staphylococcus aureus leading to oxidative stress detected by chemiluminescence while resistant strains did not suffer such stress. Oxidation of lipids was performed by employing thiobarbituric acid reaction to detect the formation of the amplified intermediate between reactive species oxygen and cytoplasmic macromolecules, namely malondialdehyde (MDA). The sensitive strain presented higher peroxidation of lipids than the resistant strain. The oxidative consequence for DNA was investigated by means of bacteria incubation with ciprofloxacin and posterior extraction of DNA, which was studied by high performance liquid chromatography (HPLC). Sensitive S. aureus ATCC 29213 showed an increase of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) respect controls without antibiotic; there was evident increase of the ratio between 8-oxodG and deoxyguanosine (dG) as a consequence of oxidation of dG to 8-oxodG considered the major DNA marker of oxidative stress. The resistant strain showed low oxidation of DNA and the analysis of 8-oxodG/dG ratio indicated lesser formation of 8-oxodG than S. aureus ATCC 29213.     

Energetic basis of cadmium toxicity in Staphylococcus aureus

In washed cells of cadmium-sensitive Staphylococcus aureus 17810S oxidizing glutamate, initial Cd²⁺⁺⁺ influx via the Mn²⁺ porter down membrane potential () was fast due to involvement of energy generated by two proton pumps—the respiratory chain and the ATP synthetase complex working in the hydrolytic direction. Such an unusual energy drain for rapid initial Cd²⁺ influx is suggested to be due to a series of toxic events elicited by Cd²⁺ accumulation down generated via the redox proton pump: (i) strong inhibition of glutamate oxidation accompanied by a decrease of electrochemical proton gradient ( _H ⁺) formation via the respiratory chain, (ii) automatic reversal of ATP synthetase from biosynthetic to hydrolytic mode, which was monitored by a decrease of _H ⁺-dependent ATP synthesis, (iii) acceleration of the initial Cd²⁺ influx down generated the reversed ATP synthetase, the alternative proton pump hydrolyzing endogenous ATP. The primary, cadmium-sensitive targets in strain 17810S seem to be dithiols located in the cytoplasmic glutamate oxidizing system, prior to the membrane-embedded NADH oxidation system. Inhibition by Cd²⁺ of _H ⁺-dependent ATP synthesis and of pH gradient (pH)-linked [¹⁴C]glutamate transport is a secondary effect due to cadmium-mediated inhibition of _H ⁺ generation at the cytoplasmic level. In washed cells of cadmium-resistant S. aureus 17810R oxidizing glutamate, Cd²⁺ accumulation was prevented due to activity of the plasmid-coded Cd²⁺ efflux system. Consequently, _H ⁺-producing and -requiring processes were not affected by Cd²⁺.     

Kinetics of the action of three selenides on Staphylococcus aureus growth as studied by microcalorimetry

The effect of three kinds of selenide on Staphylococcus aureus growth was studied by means of microcalorimetry. Differences in their capacities to inhibit the metabolism of this bacterium were observed. The rate constant k (in the log phase) in the presence of the compounds decreased with increasing concentrations of the compounds. The relationship of k and c is nearly linear for the selenium compounds. Judged from the rate constant, k, and the half-inhibitory concentration IC₅₀, the experimental results reveal that the sequence of antibiotic activity of the three tested selenides compounds is (2-hydroxy benzyl imino)ethyl n-hexyl selenide> n-butyl(2-hydroxy benzyl imino)ethyl selenide>bis[(2,4-dihydroxy benzyl imino)ethyl] selenide.     

Catalytic mechanism and cofactor preference of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus

Given the rapid rise in antibiotic resistance, including methicillin resistance in Staphylococcus aureus (MRSA), there is an urgent need to characterize novel drug targets. Enzymes of the lysine biosynthesis pathway in bacteria are examples of such targets, including dihydrodipicolinate reductase (DHDPR, E.C. 1.3.1.26), which is the product of an essential bacterial gene. DHDPR catalyzes the NAD(P)H-dependent reduction of dihydrodipicolinate (DHDP) to tetrahydrodipicolinate (THDP) in the lysine biosynthesis pathway. We show that MRSA–DHDPR exhibits a unique nucleotide specificity utilizing NADPH (K_m = 12 μM) as a cofactor more effectively than NADH (K_m = 26 μM). However, the enzyme is inhibited by high concentrations of DHDP when using NADPH as a cofactor, but not with NADH. Isothermal titration calorimetry (ITC) studies reveal that MRSA–DHDPR has ∼20-fold greater binding affinity for NADPH (K_d = 1.5 μM) relative to NADH (K_d = 29 μM). Kinetic investigations in tandem with ITC studies show that the enzyme follows a compulsory-order ternary complex mechanism; with inhibition by DHDP through the formation of a nonproductive ternary complex with NADP⁺. This work describes, for the first time, the catalytic mechanism and cofactor preference of MRSA–DHDPR, and provides insight into rational approaches to inhibiting this valid antimicrobial target.     

Detection of Methicillin-Resistant Staphylococcus aureus Using mecA/nuc Genes and Antibiotic Susceptibility Profile of Malaysian Clinical Isolates

The aim of this study is to compare methicillin-resistant Staphylococcus aureus (MRSA) detection methods and to generate antibiogram profile of S. aureus clinical isolates from two teaching hospitals in Malaysia including three reference isolates from American Type Culture Collection (ATCC). The mecA/nuc gene PCR amplification, spot inoculation test and oxacillin disc diffusion test were applied to compare its MRSA detection abilities. No disagreement between the three methods was observed. From 29 bacterial isolates (including the ATCC strains) tested, 19 isolates were confirmed as S. aureus with 14 isolates exhibiting multidrug-resistance. All isolates are still susceptible to vancomycin as indicated by the E-test result. Current biochemical tests are comparable with the molecular detection method for MRSA used in this study while multidrug-resistance traits are present in both MRSA and MSSA clinical isolates. Presently, mupirocin seems to be the best alternative for vancomycin against multidrug-resistant S. aureus infections in Malaysia. Susceptibility profile of 19 S. aureus isolates acquired from two teaching hospitals and ATCC towards 16 selected antibiotics was analyzed and an antibiogram was generated. Findings also indicated resistance against many of the available antibiotics and thus an urgent need to search for alternative antibiotics.     

Homology Modeling of Coagulase in Staphylococcus aureus

The close correlation between the ability of coagulase to clot blood plasma and their capacity to produce disease, and the corresponding absence of this property in nonpathogenic strains, have led to the assumption that the coagulase, plays important role in the pathogenesis of disease. Currently, crystal structure of coagulase in Staphylococcus aureus remains indefinable. Thus, the objectives of this research is to generate the three dimensional model of coagulase in S. aureus by using homology modeling approach. In this study, we used bioinformatics tools and databases such as BLAST (Basic Local Alignment Search Tool), GenBank, PDB (Protein Databank), and Discovery Studio to gain specific functional insights into coagulase. The model was validated using protein structure checking tools such as PROCHECK, Verify 3D and CE (Combinatorial Extension) for reliability. Therefore, structure prediction of coagulase in S. aureus can provide preliminary knowledge for understanding the function of the protein. The information from this finding will provide important information into the action and regulation mechanism of the coagulase protein in S. aureus.     

Proline betaine is a highly effective osmoprotectant for Staphylococcus aureus

Proline betaine is an osmoprotectant that is at least as effective as glycine betaine, and more effective than L-proline, for various strains of Staphylococcus aureus, and Staphylococcus epidermidis and Staphylococcus saprophyticus. ¹³C NMR studies revealed that proline betaine accumulated to high levels in osmotically stressed S. aureus, but was also detected in organisms grown in its presence in the absence of osmotic stress. Competition experiments indicated that proline betaine was taken up by the proline transport systems of S. aureus, but not by the high affinity glycine betaine transport system.Abbreviations PYK Peptode - Yeast extract K₂HPO₄     

Automatic detection of Staphylococcus aureus and Shigella dysenteriae with separated electrodes series piezoelectric sensing technique

The series piezoelectric quartz crystal (SPQC) sensing technique is a rapid and sensitive method for detection of microorganisms. In the present study, the detection device was composed of detecting system, signal generating system and data analyzing system. To magnify the amount of detection samples, eight independent SPQC sensors were parallel connected to form a muti-channel detecting unit. Electrodes were separated from the SPQC sensor and immersed into culture medium to detect the change of solution conductivity. The cell constant k was determined as 0.05 m, and the sensitivity interval of the device was from 550 to 600 μs. To maintain sensitivity of the SPQC sensor, a novel culture medium amino acid broth (AaB) was developed. It was nutrient with low conductivity and satisfied our detection device. For determining frequency detection time (FDT) expediently and accurately, FDT was defined afresh with fitting–differentiating method. Pathogens Staphylococcus aureus and Shigella dysenteriae were determined with an automated detecting device and the methods mentioned above. The calibration curves of FDT against density of bacteria showed a linear correlation coefficient (R ≥ 0.99) over the range of 10–10⁶ cells ml⁻¹. Detection results all fell inside the 95% confidence interval of a standard pour plate counting method. The reproducibility was also reviewed, and results showed that the device was stable and sensitive even after 180 days of employment.