Drug Resistance of Staphylococci

Induction of chloramphenicol (CM) resistance in Staphylococcus aureus was investigated using 166 CM derivatives and analogues. It was found that 18 compounds of the samples used were able to induce resistance to CM in Staphylococcus aureus S1477 harboring an inducible CM-resistance determinant. Most of the samples which had a nitrophenyl moiety and a D- or DL-threo isomer in its steric configuration were found to have inducer activity for CM resistance. Competent inducers are D-isomer CM derivatives which have another substituent in place of the hydroxyl group at carbon atom 3 in the propanediol of the drug.     

Drug Resistance in Staphylococcus aureus

Antibacterial and inducer activities concerning inducible macrolide resistance in Staphylococcus aureus were investigated using 32 erythromycin, oleandomycin and other macrolide antibiotic derivatives and analogues. The macrolides were classified into five groups from very high to none according to their inducer activity.     

Drug Resistance of Staphylococci

It was reported that resistance to tetracycline (TC) in some staphylococcal strains was lost irreversibly without loss of resistance to other drugs when cultured at high temperature, and that the determinant for TC resistance exists on a plasmid. According to the transductional analysis of TC resistance in Staphylococcus aureus E169 it was found that the genes which govern resistance to tetracycline and streptomycin are located close together on a single genetic element. When TC resistance in MS146, one of our stock cultures, was transduced to E169S which had lost TC resistance, TC resistance in transductants was found to be unstable at elevated temperatures. By contrast, TC resistance in transductants MS353 TC^r, to which TC resistance was transduced from E169, was indicated to be stable even when cultured at high temperature. From these results, it is strongly suggested that instability of TC resistance in E169 is not accounted for by the genetic properties of the genes which govern TC resistance but those of host cell (E169) itself.     

Bioactive Steroid Derivatives and Butyrolactone Derivatives from a Gorgonian‐Derived Aspergillus sp. Fungus

Six steroid derivatives, 1 – 6 , and five butyrolactone derivatives, 7 – 11 , were isolated from the fermentation broth of a gorgonian‐derived Aspergillus sp. fungus. Their structures were elucidated on the basis of NMR and MS spectral data. Compound 1 is a new, highly conjugated steroid. The NMR and MS data of 7 and 8 are reported for the first time, as their structures were listed in SciFinder Scholar with no associated reference. Compounds 1, 4, 5 , and 8 – 11 inhibited the larval settlement of barnacle Balanus amphitrite with EC₅₀ values ranging from 0.63 to 18.4 μg ml^?1. Butyrolactone derivatives 7 and 8 showed pronounced antibacterial activities against Staphylococcus aureus with the same MIC values as the positive control ciprofloxacin (MIC 1.56 μM for all three compounds).     

Unstable Mutants of R Factor

Thirty mutants sensitive to tetracycline were obtained from an R100 factor capable of conferring resistance to tetracycline (TC), chloramphenicol (CM), streptomycin (SM) and sulfanilamide (SA). Among the TC sensitive mutants, three showed a high frequency of spontaneous loss from host strains. The genetic loci governing the stability of R factor in host bacteria were denoted as stb. The stb^– R factors have lost many of the properties of a wild type R factor, such as, the capability to sexually transfer drug resistance and host chromosome, to confer superinfection immunity and to inhibit F function. All of these properties did not revert to a wild type phenotype, suggesting that these mutations are deletions including genetic determinants governing both TC resistance and stability of R factor. Recombinational analysis between stb^– and stb⁺ R factors indicated that crossovers between the stb loci and those governing CM (or SM.SA) resistance took place at high frequency. No crossovers were detected between stb loci and those governing TC resistance, indicating that the stb loci are linked closely to the loci governing TC resistance.     

Comparison of oxidative stress induced by ciprofloxacin and pyoverdin in bacteria and in leukocytes to evaluate toxicity

Oxidative stress induced by ciprofloxacin and pyoverdin, a leukotoxic pigment, was studied by comparing their effect in bacteria and leukocytes. Chemiluminescence (CL) assays with lucigenin or luminol were adapted to measure the stimuli of superoxide anion (O₂^?) and other reactive species of oxygen (ROS) in bacteria. Ciprofloxacin principally induced the production of O₂^? in the three species studied: Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. Lucigenin CL assay showed high oxidative stress in S. aureus due to its low superoxide dismutase (SOD) activity, whereas E. coli exhibited important SOD activity, responsible for little production of O₂^? in absence or presence of ciprofloxacin. Reduction of nitroblue of tetrazolium (NBT) was applied. This assay indicated that there was higher oxidative stress in S. aureus and E. faecalis than in E. coli. The comparison of oxidative stress generated in bacteria and leukocytes was used to check the selective toxicity of ciprofloxacin in comparison with pyoverdin. Ciprofloxacin did not generate significant stimuli of O₂^? in neutrophils, while pyoverdin duplicated the production of O₂^?. CL and NBT were useful to study the leukotoxicity of ciprofloxacin. Oxidative stress caused by the antibiotic and the leukotoxic pigment was similar in bacteria. Copyright © 2003 John Wiley & Sons, Ltd.     

Drug Resistance of Enteric Bacteria

A nontransferable R₂₁ (TC) factor was obtained by transduction of R₁₀ (TC.CM.SM.SA) with phage epsilon in group E Salmonella. The R₂₁ (TC) factor acquired transmissibility by the normal conjugal process when group E Salmonella strains harboring R₂₁ (TC) factor were infected with wild-type F or R₁₆ (CM) factor. This transmissibility at high frequency was accounted for by the formation of the recombinant F TC and R₁₀ (CM) TC factors. The F TC and R₁₆ (CM) TC factors were genetically the same as the original F and R₁₆ (CM) factors, except for the ability to confer TC resistance. In the transduction of F TC factor with phage P1, a dF TC (d: defective) factor was obtained that was defective in many F properties, such as the ability to introduce host chromosome and produce male substance, but was capable of transducing TC resistance (dF TC-infection) at low frequency.     

Exogenous application of salicylic acid alleviates cadmium toxicity and reduces hydrogen peroxide accumulation in root apoplasts of Phaseolus aureus and Vicia sativa

We examined ameliorative effects of salicylic acid (SA) on two cadmium (Cd)-stressed legume crops with different Cd tolerances, viz. Phaseolus aureus (Cd sensitive) and Vicia sativa (Cd tolerant). Cd at 50 μM significantly increased the production of hydrogen peroxide (H₂O₂) and superoxide anion (O₂^·−) in root apoplasts of P. aureus and V. sativa. When comparing the two species, we determined that Cd-induced production of H₂O₂ and O₂^·− was more pronounced in P. aureus root apoplasts than in V. sativa root apoplasts. V. sativa had higher activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) than P. aureus in root symplasts and apoplasts. Seed-soaking pretreatment with 100 μM SA decreased Cd-induced production of H₂O₂ and O₂^·− in apoplasts of both species, and increased activities of symplastic and apoplastic SOD, symplastic APX, and apoplastic CAT under Cd stress. Hence, SA-induced Cd tolerances in P. aureus and V. sativa are likely associated with increases in symplastic and apoplastic antioxidant enzyme activities.     

R Factor-Mediated Resistance to Aminoglycoside Antibiotics in Pseudomonas aeruginosa

Conjugal transferability of drug resistance was examined, in eleven Pseudomonas aeruginosa strains which were isolated in Frankfurt. Four R factors were demonstrated from three strains using P. aeruginosa as recipients but they were nontransferable to Escherichia coli K12. Two R factors, i.e., R_ms146 and R_ms147, mediated resistances to tetracycline (TC), streptomycin (SM), sulfanilamide (SA), kanamycin (KM), lividomycin (LV), gentamicin C complex (GM) and 3′,4′-dideoxykanamycin B (DKB). They mediated the formation of aminoglycoside-inactivating enzymes, i.e., SM phosphotransferase, SM adenylyltransferase, KM and LV phosphotransferase 1, and GM and DKB 6′-N-acetyltransferase. TC resistance conferred by these R factors was due to impermeability of the drug. P. aeruginosa Ps 142 carried two kinds of R factor in one cell, R_ms148 (SM) and R_ms149 (SM·SA·GM·CPC) (CPC, carbenicillin). R_ms148 (SM) was transferable at a high frequency of 10^–1 and mediated the formation of SM phosphotransferase. R_ms149 mediated the formation of drug-inactivating enzymes, i.e., GM 3-N-acetyltransferase and β-lactamase, but did not inactivate SM. SM resistance was probably due to impermeability of the drug.     

High pressure homogenization inactivation of Escherichia coli and Staphylococcus aureus in phosphate buffered saline,milk and apple juice

High pressure homogenization (HPH) offers new opportunities for food pasteurization/sterilization. Escherichia coli and Staphylococcus aureus suspended in phosphate buffered saline (PBS) buffer, milk and apple juice at initial concentration of ~10⁶ log₁₀ CFU per ml were subjected to HPH treatments up to 200 MPa with inlet temperatures at 4–40°C. After HPH at 200 MPa with the inlet temperature at 40°C, the count of E. coli suspended in PBS, milk and apple juice reduced by 3·42, 3·67 and 3·19 log₁₀ CFU per ml respectively while the count of S. aureus decreased by 2·21, 1·02 and 2·33 log₁₀ CFU per ml respectively suggesting that S. aureus was more resistant. The inactivation data were well fitted by the polynomial equation. Milk could provide a protective effect for S. aureus against HPH. After HPH at 200 MPa with the inlet temperature at 20°C, the cell structure of E. coli was destroyed, while no obvious damages were found for S. aureus.     

The effects of subinhibitory concentrations of costus oil on virulence factor production in Staphylococcus aureus

Aim: To determine the antimicrobial activity of costus (Saussurea lappa) oil against Staphylococcus aureus, and to evaluate the influence of subinhibitory concentrations of costus oil on virulence‐related exoprotein production in staph. aureus. Methods and Results: Minimal inhibitory concentrations (MICs) were determined using a broth microdilution method, and the MICs of costus oil against 32 Staph. aureus strains ranged from 0.15 to 0.6 μl ml^?1. The MIC₅₀ and MIC₉₀ were 0.3 and 0.6 μl ml^?1, respectively. Western blot, haemolytic, tumour necrosis factor (TNF) release and real‐time RT‐PCR assays were performed to evaluate the effects of subinhibitory concentrations of costus oil on virulence‐associated exoprotein production in Staph. aureus. The data presented here show that costus oil dose dependently decreased the production of α‐toxin, toxic shock syndrome toxin 1 (TSST‐1) and enterotoxins A and B in both methicillin‐sensitive Staph. aureus (MSSA) and methicillin‐resistant Staph. aureus (MRSA). Conclusion: Costus oil has potent antimicrobial activity against Staph. aureus, and the production of α‐toxin, TSST‐1 and enterotoxins A and B in Staph. aureus was decreased by costus oil. Significance and Impact of the Study: The data suggest that costus oil may deserve further investigation for its potential therapeutic value in treating Staph. aureus infections. Furthermore, costus oil could be rationally applied in food products as a novel food preservative both to inhibit the growth of Staph. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.     

Structural insights of Staphylococcus aureus FtsZ inhibitors through molecular docking, 3D-QSAR and molecular dynamics simulations

Filamentous temperature-sensitive protein Z (FtsZ) is a protein encoded by the FtsZ gene that assembles into a Z-ring at the future site of the septum of bacterial cell division. Structurally, FtsZ is a homolog of eukaryotic tubulin but has low sequence similarity; this makes it possible to obtain FtsZ inhibitors without affecting the eukaryotic cell division. Computational studies were performed on a series of substituted 3-arylalkoxybenzamide derivatives reported as inhibitors of FtsZ activity in Staphylococcus aureus. Quantitative structure-activity relationship models (QSAR) models generated showed good statistical reliability, which is evident from r²_ncv and r²_loo values. The predictive ability of these models was determined and an acceptable predictive correlation (r²_Pred) values were obtained. Finally, we performed molecular dynamics simulations in order to examine the stability of protein-ligand interactions. This facilitated us to compare free binding energies of cocrystal ligand and newly designed molecule B1. The good concordance between the docking results and comparative molecular field analysis (CoMFA)/comparative molecular similarity indices analysis (CoMSIA) contour maps afforded obliging clues for the rational modification of molecules to design more potent FtsZ inhibitors.     

Staphylococcus aureus biofilm susceptibility to small and potent β2,2-amino acid derivatives

Small antimicrobial β^2,2-amino acid derivatives (Mw < 500 Da) are reported to display high antibacterial activity against suspended Gram-positive strains combined with low hemolytic activity. In the present study, the anti-biofilm activity of six β^2,2-amino acid derivatives (A1–A6) against Staphylococcus aureus (ATCC 25923) was investigated. The derivatives displayed IC₅₀ values between 5.4 and 42.8 μM for inhibition of biofilm formation, and concentrations between 22.4 and 38.4 μM had substantial effects on preformed biofilms. The lead derivative A2 showed high killing capacity (log R), and it caused distinct ultrastructural changes in the biofilms as shown by electron and atomic force microscopy. The anti-biofilm properties of A2 was preserved under high salinity conditions. Extended screening showed also high activity of A2 against Escherichia coli (XL1 Blue) biofilms. These advantageous features together with high activity against preformed biofilms make β^2,2-amino acid derivatives a promising class of compounds for further development of anti-biofilm agents.     

Photodynamic antimicrobial activity of new porphyrin derivatives against methicillin resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Methicillin resistant Staphylococcus aureus (MRSA) with multiple drug resistance patterns is frequently isolated from skin and soft tissue infections that are involved in chronic wounds. Today, difficulties in the treatment of MRSA associated infections have led to the development of alternative approaches such as antimicrobial photodynamic therapy. This study aimed to investigate photoinactivation with cationic porphyrin derivative compounds against MRSA in in-vitro conditions. In the study, MRSA clinical isolates with different antibiotic resistance profiles were used. The newly synthesized cationic porphyrin derivatives (P_M, P_E, P_PN, and P_PL) were used as photosensitizer, and 655 nm diode laser was used as light source. Photoinactivation experiments were performed by optimizing energy doses and photosensitizer concentrations. In photoinactivation experiments with different energy densities and photosensitizer concentrations, more than 99% reduction was achieved in bacterial cell viability. No decrease in bacterial survival was observed in control groups. It was determined that there was an increase in photoinactivation efficiency by increasing the energy dose. At the energy dose of 150 J/cm² a survival reduction of over 6.33 log₁₀ was observed in each photosensitizer type. While 200 μM P_M concentration was required for this photoinactivation, 12.50 μM was sufficient for P_E, P_PN, and P_PL. In our study, antimicrobial photodynamic therapy performed with cationic porphyrin derivatives was found to have potent antimicrobial efficacy against multidrug resistant S. aureus which is frequently isolated from wound infections.     

In vitro antimicrobial activity and mechanism of action of novel carbohydrate fatty acid derivatives against Staphylococcus aureus and MRSA

Aims: This study investigates the antimicrobial activity and mode of action of novel carbohydrate fatty acid (CFA) derivatives against Staphylococcus aureus and methicillin‐resistant Staph. aureus (MRSA). Methods and Results: Minimum inhibitory concentrations (MICs) and the effect of CFA derivatives on lag phase were determined using a broth microdilution method. Lauric acid carbohydrate esters and corresponding ether analogues showed the greatest antimicrobial activity with MIC values between 0·04 and 0·16 mmol l^?1. Leakage studies at 260 nm following exposure to CFA derivatives at 4× MIC showed a significant increase in membrane permeability for all compounds, after c. 15 min exposure except for the lauric beta ether CFA derivative. Further assessment using both BacLight and luminescence ATP assays confirmed that an increase in membrane permeability and reduced metabolic activity was associated with CFA treatment. Conclusions: All strains were significantly inhibited by the novel compounds studied, and efficacy was related to specific structural features. Cell‐membrane permeabilization was associated with CFA treatment and may account for at least a component of the mode of action of these compounds. Significance and Impact of the Study: This study reports the antimicrobial action of CFA compounds against a range of Staph. aureus and MRSA strains, and provides insights into their mode of action.     

Drug Resistance of Enteric Bacteria VIII. Chromosomal Location of Nontransferable R Factor in Escherichia coli

The R₂₁(TC) factor, obtained by transduction of the R₁₀(TC.CM.SM.SA) factor with phage ε to group E Salmonella, is not transferable by the normal conjugal process. However, when R₂₁(TC)⁺ transductants are infected with the F₁₃ factor, the nontransferable R₂₁(TC) factor acquires transmissibility by conjugation. R₂₁(TC)⁺ conjugants of Escherichia coli K-12, to which only the R₂₁(TC) factor was transmitted by cell-to-cell contact from an F′ R⁺ donor, were still unable to transfer their R₂₁(TC) factor by conjugation. In crosses between Hfr and F⁻E. coli K-12 strains containing R₂₁(TC), the gene responsible for tetracycline resistance was located on the E. coli K-12 chromosome between lac and pro, near lac.     

A sensitive fluorescence method for detection of E. Coli using rhodamine 6G dyeing

Negatively charged bacteria combined with positively charged alkaline dye rhodamine 6G (Rh6G) in NaH₂PO₄–Na₂HPO₄ buffer solution pH 7.4, by electrostatic interaction. The dyed bacteria exhibited a strong fluorescence peak at 552 nm and fluorescence intensity was directly linear to Escherichia coli (E. coli), Bacillus subtilis (B. subtilis) and Staphylococcus aureus (S. aureus) concentrations in the range of 7.06 × 10⁴ to 3.53 × 10⁷, 4.95 × 10⁵ to 2.475 × 10⁸ and 32.5 to 16250 colony forming unit/mL (cfu/mL) respectively, with detection limits of 3.2 × 10⁴ cfu/mL E. coli, 2.3 × 10⁵ cfu/mL B. subtilis and 16 cfu/mL S. aureus, respectively. Samples were cultured for 12 h, after which the linear detection range for E. coli was 2 to 88 cfu/mL. This simple, rapid and sensitive method was used for the analysis of water and drinking samples. Copyright © 2015 John Wiley & Sons, Ltd.     

QTL mapping for European corn borer resistance (<Emphasis Type="Italic">Ostrinia nubilalis</Emphasis> Hb.), agronomic and forage quality traits of testcross progenies in early-maturing European maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) germplasm

In hybrid breeding the performance of lines in hybrid combinations is more important than their performance per se. Little information is available on the correlation between individual line and testcross (TC) performances for the resistance to European corn borer (ECB, Ostrinia nubilalis Hb.) in maize (Zea mays L.). Marker assisted selection (MAS) will be successful only if quantitative trait loci (QTL) found in F₂ derived lines for ECB resistance are still expressed in hybrid combinations. The objectives of our study were: (1) to identify and characterize QTL for ECB resistance as well as agronomic and forage quality traits in a population of testcrossed F_2:3 families; (2) to evaluate the consistency of QTL for per se and TC performances; and (3) to determine the association between per se and TC performances of F_2:3 lines for these traits. Two hundred and four F_2:3 lines were derived from the cross between maize lines D06 (resistant) and D408 (susceptible). These lines were crossed to D171 and the TC progenies were evaluated for ECB resistance and agronomic performance in two locations in 2000 and 2001. Using these TC progenies, six QTL for stalk damage rating (SDR) were found. These QTL explained 27.4% of the genotypic variance in a simultaneous fit. Three QTL for SDR were detected consistently for per se and TC performance. Phenotypic and genotypic correlations were low for per se and TC performance for SDR. Correlations between SDR and quality traits were not significant. Based on these results, we conclude that MAS will not be an efficient method for improving SDR. However, new molecular tools might provide the opportunity to use QTL data as a first step to identify genes involved in ECB resistance. Efficient MAS procedures might then be based on markers designed to trace and to combine specific genes and their alleles in elite maize breeding germplasm.Communicated by G. Wenzel     

Antimicrobial efficacy of lactoferrin, its amidated and pepsin-digested derivatives, and their combinations, on Escherichia coli O157:H7 and Serratia liquefaciens

Aims: To evaluate the in vitro bactericidal efficacy of lactoferrin (LF), its amidated (AMILF) and pepsin‐digested (PDLF) derivatives, and their combinations, on Escherichia coli O157:H7 and Serratia liquefaciens. Methods and Results: PDLF exhibited the most potent bactericidal efficacy on E. coli O157:H7 (>2·5 log₁₀ CFU ml^?1 reduction at concentrations ≥1 mg ml^?1), and AMILF on Ser. liquefaciens (1 log₁₀ CFU ml^?1 reduction at 0·25–0·50 mg ml^?1). Some combinations of LF with PDLF or AMILF showed a slight synergy on E. coli O157:H7 and Ser. liquefaciens. However, all combinations of AMILF with PDLF were less active than the sum of the individual effects of the two antimicrobials. Production of capsular polysaccharide by bacteria might be involved in antimicrobial resistance. Conclusions: Escherichia coli O157:H7 and Ser. liquefaciens showed marked differences in the sensitivity to LF and its derivatives. E. coli O157:H7 was strongly inhibited by PDLF, whereas the effect of LF and its derivatives on Ser. liquefaciens was weak to negligible. Significance and Impact of the Study: PDLF was the most promising of the tested antimicrobials on E. coli O157:H7. However, the resistance of Ser. liquefaciens to LF and its derivatives hinders their use in the food industry.