Kanamycin films for ulcerative lesions of the cornea]

The kanamycin films were used once for the treatment of 28 patients with the cornea ulcers. The positive effect was registered in 20 patients. Eight patients were treated with the films and the antibiotic acqueous solution. The best results were observed in the patients with affection of the surface layers of the cornea. The positive therapeutic results were registered in 20 patients after operations in connection with cataracta and glaucoma when treated with the films containing kanamycin and dexametazon.     

A Staphylococcus aureus mouse keratitis topical infection model: cytokine balance in different strains of mice

Staphylococcus is a leading cause of the potentially blinding disease microbial keratitis. Even with the use of antibiotic therapy, the host inflammatory response continues to damage the cornea, which may lead to blindness. Manipulation of the host response may help improve patient outcome from this devastating disease. We aim to understand the contribution of the host response to Staphylococcus aureus infection. A S. aureus keratitis mouse model was developed in both C57BL/6 and BALB/c mice using two different strains of S. aureus (8325-4 and Staph 38). Twenty-four hours postinfection, mice were killed and eyes were harvested for enumeration of bacteria, polymorphonuclear leucocytes, chemokines and cytokines. The laboratory strain 8325-4 was not as virulent as the clinical isolate Staph 38. In vitro data showed a 250-fold increase in invasion of human corneal epithelial cells by Staph 38 compared to 8325-4. BALB/c mice were susceptible to S. aureus infection whereas C57BL/6 mice were resistant. The resistant C57BL/6 mice were polarized towards a Th2 response, which may be protective for these mice. IL-4, IL-10 and IL-6 were elevated significantly in C57BL/6 mice infected with Staph 38 (P < 0.05). Macrophage inflammatory peptide (MIP)-2 was also significantly elevated in C57BL/6 mice (P < 0.001). The susceptible BALB/c mice had a muted cytokine response, which suggests that S. aureus might be 'walled off' during infection and might avoid host defences. IL-4, IL-10 and IL-6 cytokines may be protective during Gram-positive corneal infection and therefore may be useful for adjunct therapies in the treatment of this disease.     

Mupirocin resistance in staphylococci: development and transfer of isoleucyl-tRNA synthetase-mediated resistance in vitro

Mupirocin resistance could be transferred from highly resistant clinical isolates of Staphylococcus aureus to highly sensitive recipients of Staph. aureus, Staph. epidermidis and Staph. haemolyticus. Transconjugants of the latter two organisms could transfer this resistance into mupirocin-sensitive Staph. aureus. Moderately resistant strains did not transfer this resistance to sensitive recipients, nor did strains with high-level mupirocin resistance developed by serial transfer or habituation. The inhibitory effects of mupirocin on crude isoleucyl-tRNA synthetases (IRS) isolated from mupirocin-sensitive and -resistant strains of Staph. aureus have been determined. Drug concentrations needed to produce 50% inhibition, I50 values, were very low against IRS from a highly sensitive strain, somewhat higher against IRS from moderately resistant strains, much higher against enzyme from strains trained in vitro to high-level resistance, and considerably higher still against IRS extracted from clinical isolates possessing high-level mupirocin resistance and from the transconjugates of such strains resulting from crosses with mupirocin-sensitive strains. It is concluded that high-level resistance in clinical isolates is plasmid-mediated involving a second, mupirocin-resistant IRS whereas in moderately resistant strains, and in strains trained in vitro to high-level resistance, chromosomal mutations are likely to be responsible for decreasing IRS sensitivity.     

Hazard analysis applied to microbial growth in foods: development of mathematical models describing the effect of water activity

Mathematical models have been developed which describe the effect of lowering the water activity on the growth kinetics of Staphylococcus aureus and Salmonella typhimurium. By treating the lag phase and exponential phase kinetics separately predictions can be made on the extent of microbial growth over successive time/temperature cycles. Staph. aureus was far more tolerant than Salm. typhimurium to lowered water activity and under near growth limiting conditions of water activity and temperature was showing lag periods as long as ca 40 d. The maximum lag period observed for Salm. typhimurium was ca 5 d. Under these conditions the predicted generation times for Staph. aureus were 2-3 d and for Salm. typhimurium.     

THE DISINFECTANT PROPERTIES OF QUATERNARY AMMONIUM COMPOUNDS AND SODIUM HYPOCHLORITE: A COMPARISON OF RESULTS USING DIFFERENT TEST METHODS

SUMMARY: On exposing a strain of Bacterium coli 28.D.10 in a surface film at atmospheric temperature to atmospheres of different moisture contents, it was found that for relative humidities between 100 and 66% the numbers of survivors decreased with decreasing humidity. There was also some evidence of a slight increase in survivors for a decrease in relative humidity from 43 to 0%.
The percentage of survivors of Bact. coli after exposure to quaternary ammonium disinfectants also decreased with relative humidity between 100 and 66% but no significant differences were found for changes in relative humidity below 66%. The numbers of survivors of a culture of Staphylococcus aureus were the same after storage at a relative humidity of 43% as at 100%; drying did not appear to affect the sensitivity of Staph. aureus to quaternary ammonium compounds. Tests of the effect of storage time in a saturated atmosphere gave results which were not entirely consistent, but where differences were observed, there was a lower percentage of survivors for freshly inoculated films than for films which had been stored for 3 hr, presumably because a fresh film was more easily removed to the disinfectant.
When either Bact. coli or Staph. aureus was exposed to a disinfectant, the percentage of survivors was higher when the organisms were in a surface film than when they were inoculated directly into the disinfectant. Agitation during exposure reduced the numbers of survivors from a surface film. Neither the glass nor the metal coming in contact with the disinfectants affected the level of survivors.
Under the conditions of testing, sodium hypochlorite was a more effective disinfectant than the quaternary ammonium compounds used.     

Hazard analysis applied to microbial growth in foods: Development of mathematical models describing the effect of water activity

Mathematical models have been developed which describe the effect of lowering the water activity on the growth kinetics of Staphylococcus aureus and Salmonella typhimurium. By treating the lag phase and exponential phase kinetics separately predictions can be made on the extent of microbial growth over successive time-temperature cycles. Staph. aureus was far more tolerant than Salm. typhimurium to lowered water activity and under near growth limiting conditions of water activity and temperature was showing lag periods as long as ca 40 d. The maximum lag period observed for Salm. typhimurium was ca 5 d. Under these conditions the predicted generation times for Staph. aureus were 2–3 d and for Salm. typhimurium 1–4 d.     

Immunomodulating action of myelopeptides in severe closed experimental craniocerebral injury in rats

An immunocorrective effect of myelopeptides (MP) isolated from pig bone marrow cell culture supernatant in the early posttraumatic period in rats with severe cranial injury has been assessed. MP administration prevented cellular devastation of thymus and bone marrow, as well as spleen hyperplasia. The most marked MP effect was observed within the first 24 h after its administration. MP affected the functional and migration properties of both the entire population of lymphoid cells and individual subpopulations. MP had a pronounced protective effect against Staph. aureus infection during cranial trauma. MP completely prevented the death of animals and reduced more than 3-fold Staph. aureus persistence in the organism of animals. Anti-stress and protective effects of MP open vast prospects for their therapeutic and preventive application in the clinical practice.     

Antibiotic activity of isoxazolylnaphthoquinone imines on mice infected with Staphylococcus aureus

I. ALBESA, P. BOGDANOV, A. ERASO, N.R. SPERANDEO AND M.M. DE BERTORELLO. 1995. The antibiotic activity of new synthetic isoxazolylnaphthoquinone imines was studied. Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were resistant to the four compounds studied (MIC > 128 µg ml⁻¹), but Staphylococcus aureus ATCC 25923, ATCC 29213 and 30 clinical isolates of Staph. aureus were inhibited by 2-hydroxy- N -(3,4-dimethyl-5-isoxazolyl)-1,4-naphthoquinone-4-imine (I). This compound diminished bloodstream infection of mice injected i.m. with Staph. aureus; septicaemia decayed significantly when I was applied at the beginning of the infection while when I was given 3 d after bacterial challenge, a significant protection was afforded. Bactericidal activity in serum increased during the 5 h after I was administered i.p.
The acetyl derivative of I had a high MIC but when inoculated orally in mice decreased the Staph. aureus counts in circulation. This protection occurred only when the schedule of administration started close to the bacterial challenge. Antibiotic activity in vivo may be associated with in vitro effects.     

Flora of the purulent foci of patients at an orthopedic traumatology clinic and its sensitivity to antibiotics

A total of 4664 bacteriological analyses of the wound and purulent discharge from orthopedo-traumatological patients were performed within a 6-year period, i.e. from 1971 to 1976. Staphylococci were the dominating microbes of the purulent-inflammatory foci. Its part in the monoculture amounted to 64.5--82 per cent. The specific weight of the monocultures of various microbes decreased during the last 3 years, while the number of the microbial associations increased from 11.6 to 25.4 per cent Staph. aureus predominated in the inflammatory processes (65.8 to 86.5 per cent). Still, during the last 3 years the number of Staph. epidermidis increased from 16.8 to 26.2 per cent. The number of the so called "intermediate" or dissociated type of Staphylococcus, i.e. Staph. albus usually amounted to 7.5--8.1 per cent. In 1976 its number was 12.5 per cent. The pathogenic microbes of the coccal group were usually sensitive to erythromycin, monomycin, levomycetin and kanamycin. Among these microbes only staphylococci preserved their sensitivity to penicillin. The causative agents of purulent processes, i.e. Escherichia, Proteus, Pseudomonas were resistant to most of the antibiotics. Sensitivity to monomycin was preserved by 50 per cent only in Proteus. The microbial associations were mainly sensitive to monomycin and kanamycin.     

Rapid detection of enterotoxigenic Staphylococcus aureus harbouring genes for four classical enterotoxins, SEA, SEB, SEC and SED, by loop-mediated isothermal amplification assay

AIM: The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay targeting the genes for the four classical enterotoxins, SEA, SEB, SEC and SED, in Staphylococcus aureus. METHODS AND RESULTS: Specific primers were designed which target each specific sequence of the enterotoxin genes. With 30 strains of Staph. aureus, the results of the LAMP assay to each enterotoxin, SEA, SEB, SEC and SED, completely accorded with the results of polymerase chain reaction (PCR) assay. Enterotoxin production, determined by a reverse passive latex agglutination assay, strongly correlated with the presence of the corresponding genes. Amplification was not observed when 14 strains of nonenterotoxigenic Staph. aureus and 20 strains consisting of 19 bacterial species other than Staph. aureus were tested. In addition, the sensitivity of the LAMP assay was generally higher than that of conventional PCR assay and it rapidly detected enterotoxigenic Staph. aureus strains within 60 min. CONCLUSIONS: The LAMP assay developed in this study is rapid, specific and sensitive for the detection of enterotoxigenic Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is suitable for clinical diagnosis and food safety applications.     

Accumulation of ppGpp and ppGp in Staphylococcus aureus 8325-4 following nutrient starvation

AIM: To investigate the accumulation of highly phosphorylated guanosine nucleotides in Staphylococcus aureus 8325-4 following nutrient deprivation. METHODS AND RESULTS: Nutrient shiftdown of Staph. aureus, HPLC of nucleotides and Western blotting of cell-free extracts. ppGpp rapidly accumulated when cells were deprived of isoleucine following addition of mupirocin, or after carbon deprivation. In contrast, total amino acid starvation led to delayed production of ppGp, which suggests that Staph. aureus exhibits a unique response to total amino acid deprivation compared with other eubacteria. Intracellular ppGp was observed at high levels under all starvation conditions, which suggests that this nucleotide is linked to nutrient limitation and may therefore be involved in regulating the stringent response in Staph. aureus. pppGpp was not observed under any nutrient-limiting condition. Western blot analysis of whole-cell extracts from Staph. aureus 8325-4, showed that antibodies to RelA and SpoT cross-reacted under conditions that detected these proteins in Escherichia coli. CONCLUSIONS: Staph. aureus produces ppGpp and ppGp following nutrient limitation. Immunological analysis indicates that Staph. aureus contains RelA and SpoT proteins, similar to those produced by E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a new example of the diversity of metabolic regulations in bacteria.     

Combination of novoimanine with antibiotics with a different mechanism of action]

Ampicillin, kanamycin, fusidic acid and rifocin significantly increased the effect of novoimanin on Staph. aureus 209. Histon F1 and spectinomycin did not influence the effect of novoimanin. Inefficiency of novoimanin combination with histon F1 provided a supposition that their effect may be directed to the same cell structures and most probably to the membranes. This was confirmed by the data of electron microscopy. The most effective combinations were recommended for the studies on their possible clinical use.     

Effect of Pseudomonas aeruginosa melanin on antibiotic activity]

The properties of microbial melanines are very diverse. Melanine of P. aeruginosa is little studied. The pigment was isolated from a strain of P. aeruginosa possessing all characteristic properties of the species. Interaction of P. aeruginosa melanine with various antibiotics was determined by the method of serial dilutions in beaf-peptone broth, using Staph. aureus 209 as a test-microbe, which was added to the medium in an amount of 10(6) cells to each tube. It was found that P. aeruginosa melanine differed from DOPA-melanine in a concentration of 1 mg/ml and did not change the activity of penicillin, tetracycline, oleandomycin, kanamycin and gentamicin with respect to Staph. aureus.     

Rapid detection of Staphylococcus aureus using bioluminescent enzyme immunoassay

A bioluminescent enzyme immunoassay (BLEIA) method for detecting protein A-bearing Staphylococcus aureus was developed using biotinylated firefly luciferase. The BLEIA was able to detect protein A at one pg ml-1 and 103 cfu ml-1 level of Staph. aureus. The BLEIA showed significant signals with overnight cultures of all 24 Staph. aureus strains, and the BLEIA did not show any significant signals with overnight cultures of all 44 strains of coagulase-negative staphylococci and the other genus bacteria. After 5 h cultivation beginning at approximately 50 cfu ml-1, the BLEIA was able to detect all 35 Staph. aureus strains isolated from healthy humans.     

Detection of Staphylococcus aureus and enterotoxin genotype diversity in Monte Veronese, a Protected Designation of Origin Italian cheese

AIMS: To evaluate the risk associated with the load and enterotoxigenicity of Staphylococcus aureus in Monte Veronese, a PDO (Protected Designation of Origin) cheese of the Lessinia area (Verona, Italy). METHODS AND RESULTS: Staphylococcus aureus was quantified by a conventional culture method and by a nucA targeted real-time PCR assay developed in this study. Staphylococcus aureus numbers in cheese were higher than the limit tolerated by the Italian food legislation in 78% instances, according to both detection methods. Multiplex PCR tests for 17 Staph. aureus enterotoxin (SE) genes were applied to nucleic acids extracted from curds, cheeses and Staph. aureus isolates. The SE gene diversity appeared reduced after ripening. The gene encoding SED was found most frequently in dairy samples and the enterotoxin genes ser, sed, seg and sem predominated in the isolates. CONCLUSIONS: The occurrence of enterotoxigenic Staph. aureus strains with complex SE genotypes in this PDO cheese at numbers often exceeding the Italian tolerance threshold represents an important risk factor. SIGNIFICANCE AND IMPACT OF THE STUDY: The high frequency of contamination of Monte Veronese PDO cheese and, expectedly, similar typical productions from raw milk, by enterotoxigenic Staph. aureus imposes a tighter hygienic control in the earlier manufacturing phases.     

Direct detection of nasal Staphylococcus aureus carriage via helicase-dependent isothermal amplification and chip hybridization

ABSTRACT: BACKGROUND: The bacterium Staphylococcus aureus constitutes one of the most important causes of nosocomial infections. One out of every three individuals naturally carries S. aureus in their anterior nares, and nasal carriage is associated with a significantly higher infection rate in hospital settings. Nasal carriage can be either persistent or intermittent, and it is the persistent carriers who, as a group, are at the highest risk of infection and who have the highest nasal S. aureus cell counts. Prophylactic decolonization of S. aureus from patients' noses is known to reduce the incidence of postsurgical infections, and there is a clear rationale for rapid identification of nasal S. aureus carriers among hospital patients. FINDINGS: A molecular diagnostic assay was developed which is based on helicase-dependent target amplification and amplicon detection by chip hybridization to a chip surface, producing a visible readout. Nasal swabs from 70 subjects were used to compare the molecular assay against culturing on "CHROMagar Staph aureus" agar plates. The overall relative sensitivity was 89%, and the relative specificity was 94%. The sensitivity rose to 100% when excluding low-count subjects (<100 S. aureus colony-forming units per swab). CONCLUSIONS: This molecular assay is much faster than direct culture and has sensitivity that is appropriate for identification of high-count (>100 S. aureus colony-forming units per swab) nasal S. aureus carriers who are at greatest risk for nosocomial infections.     

The inhibition of Staphylococcus hyicus on a selective medium for staphylococci

Growth of Staphylococcus hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains was found to be severely inhibited when broth cultures of these organisms were streaked on Schleifer and Kramer's staphylococcal (SK) medium. Of the selective agents contained in SK medium, potassium thiocyanate was found to be inhibitory towards both subspecies of Staph. hyicus and sodium azide had an additional inhibitory effect on Staph. hyicus subsp. chromogenes. Of six different media supplements examined, sheep blood and Tween 80 were found to improve the growth of both Staph. hyicus subspecies when added to SK medium. These findings were confirmed in subsequent work where the supplemented SK media were used to isolate potential enterotoxin-producing organisms from simulated raw milk (Staph. aureus, Staph. hyicus subsp. hyicus, Staph. hyicus subsp. chromogenes, Staph. intermedius ). SK medium supplemented with sheep blood proved more effective in allowing satisfactory recovery of Staph. aureus, Staph. hyicus subsp. hyicus and Staph. intermedius. However, neither supplement enabled satisfactory recovery of Staph. hyicus subsp. chromogenes to be achieved.     

The inhibition of Staphylococcus hyicus on a selective medium for staphylococci

Growth of Staphylococcus hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains was found to be severely inhibited when broth cultures of these organisms were streaked on Schleifer and Kramer's staphylococcal (SK) medium. Of the selective agents contained in SK medium, potassium thiocyanate was found to be inhibitory towards both subspecies of Staph. hyicus and sodium azide had an additional inhibitory effect on Staph. hyicus subsp. chromogenes. Of six different media supplements examined, sheep blood and Tween 80 were found to improve the growth of both Staph. hyicus subspecies when added to SK medium. These findings were confirmed in subsequent work where the supplemented SK media were used to isolate potential enterotoxin-producing organisms from simulated raw milk (Staph. aureus, Staph. hyicus subsp. hyicus, Staph. hyicus subsp. chromogenes, Staph. intermedius). SK medium supplemented with sheep blood proved more effective in allowing satisfactory recovery of Staph. aureus, Staph. hyicus subsp. hyicus and Staph. intermedius. However, neither supplement enabled satisfactory recovery of Staph. hyicus subsp. chromogenes to be achieved.     

Properties of Staphylococcus aureus in case of the unfavorable course of burn infection

Clinical and microbiological study of wound discharge from 35 patients demonstrated a relationship between biological properties of S. aureus, the causative agent of burn infection, and the course of the infected burn trauma. The prognostic importance of the antioxidant properties of these staphylococci was established: in cases of the unfavorable course of the burn process they showed essentially greater resistance to peroxinitrite and higher superoxide dismutase activity in comparison with the cultures isolated from patients with uncomplicated wound infection.