Effect of detergents on the chloramphenicol inactivation process by resistent bacteria

The effect of detergents, i. e. cationic, anionic, nonionic and polyelectrolytes of the cationic type on the efficacy of chloramphenicol against resistant strains of E. coli and Staph. aureus was studied. It was found that the detergent effect on inactivation of chloramphenicol by the bacterial resistant strains was inconsistent. The cationic detergents and in particular chlorhexidine had the most pronounced inhibitory effect. In subbacteriostatic concentrations they significantly suppressed inactivation of chloramphenicol in the cells of E. coli and Staph. aureus. The anionic detergents and polyelectrolytes of the cationic type in the above concentrations were effective only with respect to Staph. aureus. It is noted that the detergents increased the activity of chloramphenicol against E. coli and Staph. aureus.     

Variation in the behaviour of enterotoxigenic Staphylococcus aureus after heat stress in milk

The survival of several strains of Staphylococcus aureus after heat stress in different menstrua was not logarithmic and F-values were determined to express their resistance to heat. Of the strains tested, Staph, aureus 234 (enterotoxin B) was the most heat resistant and Staph. aureus 790 (enterotoxin E) was the most heat sensitive. Buffalo milk gave the best protection to all the strains of Staph. aureus against heat, followed by cow's milk; phosphate-buffered saline gave the least protection. Soyabean casein digest agar gave maximum recovery of survivors followed by brain heart infusion and Baird-Parker medium. At 50°C there was no marked variation in coagulase production by the surviving strains but at 55 and 62–5dE C there was complete loss of coagulase activity. There was a decreased deoxyribonuclease (DNase) production by all the strains of Staph. aureus after heat stress. Heat-treatment at 55 and 62mD5dE C resulted in loss of enterotoxin production by all the survivors except S₆ and 234, the surviving cells of which still prodused enterotoxin B after heat treatment at 55dE C. Most of the survivors regained lost characteristics such as coagulase, DNase and enterotoxin production after four to five passages through BHI which suggests that subculture of Staph. aureus recovered from heat-processed milk is necessary to avoid false results.     

Determination of susceptibility of Staphylococcus aureus to methicillin by luciferim-luciferase assay of bacterial adenosine triphosphate

M c W alter , P.W. 1984. Determination of susceptibility of Staphylococcus aureus to methicillin by luciferin-luciferase assay of bacterial adenosine triphosphate. Journal of Applied Bacteriology , 56 , 145–150.
Susceptibility of 50 strains of Staphylococcus aureus to methicillin was determined by disc diffusion, minimum inhibitory concentration (MIC) test and luciferin-luciferase assay of bacterial adenosine triphosphate (ATP). ATP concentration time curves calculated on eight strains (four sensitive, four resistant) incubated at 30C indicated 2.5 h as the optimum time for determination of methicillin susceptibility. The ATP concentration and relative light unit (RLU) value of each test broth (antibiotic-containing), expressed as a percentage of its own control broth (antibiotic-free) indicated values of < 30% for ATP and < 40% for RLU to be indicative of methicillin sensitivity. Single time point ATP assays carried out on the 50 strains after 2.5 h at 30C correlated exactly with disc diffusion and MIC. Luciferin-luciferase assay of bacterial ATP appeared to be a reliable, rapid technique for determining the susceptibility of Staph. aureus to methicillin.     

Protective Efficacy of Passive Immunization with Monoclonal Antibodies in Animal Models of H5N1 Highly Pathogenic Avian Influenza Virus Infection

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.     

Variation in the behaviour of enterotoxigenic Staphylococcus aureus after heat stress in milk

The survival of several strains of Staphylococcus aureus after heat stress in different menstrua was not logarithmic and F-values were determined to express their resistance to heat. Of the strains tested, Staph. aureus 234 (enterotoxin B) was the most heat resistant and Staph. aureus 790 (enterotoxin E) was the most heat sensitive. Buffalo milk gave the best protection to all the strains of Staph. aureus against heat, followed by cow's milk; phosphate-buffered saline gave the least protection. Soyabean casein digest agar gave maximum recovery of survivors followed by brain heart infusion and Baird-Parker medium. At 50 degrees C there was no marked variation in coagulase production by the surviving strains but at 55 and 62.5 degrees C there was complete loss of coagulase activity. There was a decreased deoxyribonuclease (DNase) production by all the strains of Staph. aureus after heat stress. Heat-treatment at 55 and 62.5 degrees C resulted in loss of enterotoxin production by all the survivors except S6 and 234, the surviving cells of which still produced enterotoxin B after heat treatment at 55 degrees C. Most of the survivors regained lost characteristics such as coagulase, DNase and enterotoxin production after four to five passages through BHI which suggests that subculture of Staph. aureus recovered from heat-processed milk is necessary to avoid false results.     

The role of gamma interferon in acquired host resistance against Staphylococcus aureus infection in mice

We investigated the expression of an acquired host resistance against Staphylococcus aureus infection in mice. When C57BL/6 mice were immunized with viable S. aureus and challenged with S. aureus eight weeks later, the elimination of S. aureus from the spleen and liver was enhanced in the immunized mice compared with the nonimmunized mice. When gamma interferon (IFN-gamma(-/-)) mice were immunized and challenged, the bacterial numbers in the organs of immunized mice were comparable to those in the nonimmunized mice, suggesting that IFN-gamma plays a critical role in an acquired host resistance against S. aureus infection. IFN-gamma(-/-) mice produced the lower level of anti-S. aureus immunoglobulin M (IgM) and IgG2a antibodies compared with C57BL/6 mice. To elucidate the role of IFN-gamma produced during a challenge with S. aureus, a single injection of anti-IFN-gamma monoclonal antibody to mice was carried out 1 h before challenge. An acquired resistance against S. aureus infection was inhibited by injecting with anti-IFN-gamma monoclonal antibody. However, anti-IFN-gamma monoclonal antibody treatment failed to modulate anti-S. aureus IgM, IgG1 or IgG2a responses in these animals. These results demonstrated that IFN-gamma is required for an acquired resistance against S. aureus infection in mice. However, IFN-gamma induced during the challenge failed to affect the secondary antibody responses.     

Therapeutic effect of some antibiotics on experimental staphylococcal infection and its correlation with in vitro activity of antibiotics in sub-inhibitory concentration against Staphylococcus aureus strains

The aim of the study was to demonstrate of whether the therapeutic effects of antibiotics depend on their in vitro activity in sub-inhibitory concentrations against staphylococci. Cloxacillin, gentamicin and lincomycin were used in the study. Groups of S. aureus strains, containing 6 strains with similar MIC values each but different sensitivity to sub-inhibitory antibiotic concentrations (sub-MIC) were selected (a total of 36 trains): i. strains increasing their sensitivity to phagocytosis and bactericidal activity of rabbit leukocytes after incubation with an antibiotic in 0.1 MIC concentration, ii. strains with sensitivity to the above factors unaffected by incubation with an antibiotic in 0.5 MIC concentration. The doses of staphylococci causing death of 90-100% of Swiss albino mice 10 days after i.p. infection were determined. The injected doses (LD 90-100) and various doses of antibiotics were used to determine ED50 values as well as the survival rate of the mice with experimental staphylococcal infections after treatment with these antibiotics. It was demonstrated that effective doses (ED 50) of the antiboitics were significantly lower when the antibiotics were administered once to mice infected with strains S. aureus sensitive to sub-MIC concentrations of the investigated antibiotics than for mice infected with strains resistant to their sub-MIC concentrations. Similar correlations were observed in mice which were given the antibiotics several times (for 7 days): the percentage of the surviving mice was higher in the group infected with sub-MIC sensitive strains. The therapeutic effect of cloxacillin, gentamicin and lincomycin demonstrated a significant correlation with the S. aureus strains used to induce the infections and their sensitivity, or lack of sensitivity in vitro, to phagocytosis and bactericdal activity of leukocytes in the presence of antibiotics in sub-MIC concentrations.     

Intranasal vaccination with a double mutant of staphylococcal enterotoxin C provides protection against Staphylococcus aureus infection

Staphylococcus aureus expresses a repertoire of factors including staphylococcal exotoxins (SEs), exoenzymes, and numerous cell-associated components that contribute to the pathogenesis of disease. We constructed and expressed a nontoxic double mutant SEC (dmSEC), devoid of superantigenic activity, and investigated the ability of intranasal vaccination with dmSEC plus cholera toxin (CT) adjuvant to protect mice against S. aureus infection. Mice were vaccinated with dmSEC and inoculated with a viable S. aureus clinical isolate strain. The survival rate in the immunized mice was higher, and bacterial counts in the organs were significantly lower than those in the control group. Intranasal vaccination with dmSEC induced the production of SEC-specific antibodies such as IgG1, IgG2b and IgA. dmSEC-vaccinated mice elicited significantly higher titers of interleukin-4 (IL-4) and IL-10, and lower levels of interferon-gamma (IFN-gamma) after challenge with S. aureus compared with the control group. Furthermore, the sera from dmSEC-immunized mice significantly inhibited IFN-gamma and tumor necrosis factor-alpha production in vitro. These results indicate that intranasal vaccination with dmSEC devoid of superantigenic properties induces systemic immune responses and provides protection against S. aureus infection.     

Influenza A inhibits Th17-mediated host defense against bacterial pneumonia in mice

Staphylococcus aureus is a significant cause of hospital and community acquired pneumonia and causes secondary infection after influenza A. Recently, patients with hyper-IgE syndrome, who often present with S. aureus infections of the lung and skin, were found to have mutations in STAT3, required for Th17 immunity, suggesting a potential critical role for Th17 cells in S. aureus pneumonia. Indeed, IL-17R(-/-) and IL-22(-/-) mice displayed impaired bacterial clearance of S. aureus compared with that of wild-type mice. Mice challenged with influenza A PR/8/34 H1N1 and subsequently with S. aureus had increased inflammation and decreased clearance of both virus and bacteria. Coinfection resulted in greater type I and II IFN production in the lung compared with that with virus infection alone. Importantly, influenza A coinfection resulted in substantially decreased IL-17, IL-22, and IL-23 production after S. aureus infection. The decrease in S. aureus-induced IL-17, IL-22, and IL-23 was independent of type II IFN but required type I IFN production in influenza A-infected mice. Furthermore, overexpression of IL-23 in influenza A, S. aureus-coinfected mice rescued the induction of IL-17 and IL-22 and markedly improved bacterial clearance. These data indicate a novel mechanism by which influenza A-induced type I IFNs inhibit Th17 immunity and increase susceptibility to secondary bacterial pneumonia.     

Influence of lactoferrin feeding and injection against systemic staphylococcal infections in mice

Human and bovine lactoferrins (Lfs) and bovine lactoferrin hydrolysate (LH) were assessed in vitro and in vivo for their antibacterial effects on Staphylococcus aureus. Lactoferrins showed weak in vitro antibacterial activity while Fe-saturated Lfs and LH showed no activity. Lactoferrin-treated mice (1 mg, i.v.) when injected i.v. with 10(6) staphylococci, showed 30-50% reduction in kidney infections, and viable bacterial counts in the kidneys decreased 5-12-fold. The inhibitory effect was dose-dependent up to 1 mg Lf. Lactoferrins were effective when given 1 day prior to the bacterial challenge, after which there was no significant effect even at doses up to 5 mg. Apo- and Fe-saturated forms of human and bovine Lfs were all equally effective, while LH was not protective. Human and bovine Lfs with different degrees of iron saturation (9-97%) were found to be equipotent. Feeding mice with 2% bLf in drinking water also reduced the kidney infections by 40-60%, and viable bacterial counts, 5-12-fold. The results suggest a potential for the use of Lfs as natural antibacterial proteins for preventing bacterial infections.     

Feasibility of prophylaxis and therapy against Gram-negative infections by human monoclonal antibodies

Abstract In a murine model of Gram-negative sepsis, we have shown that the prophylactic application of human monoclonal antibodies (HmAbs) with specificity for lipopolysaccharides (LPS) of Pseudomonas aeruginosa protected against bacterial infection. In this paper we show that the therapeutical application of 5 μg of these HmAbs up to 6 h after challenge with a lethal dose of live P. aeruginosa results in a protection rate of 70–90%. Administration 18 h after bacterial challenge, diminished the protection to 43% survival rate. Furthermore, using a mixture of HmAbs recognizing a total of six different P. aeruginosa serotypes, no interference in their protective capacities was found. Finally, these HmAbs also protected galactosamine-sensitized mice against lethal challenge with LPS. Our data show that the described HmAbs confer bactericidal activity as well as anti-endotoxic activity in vivo.     

Minimum inhibitory concentration of smoke wood extracts against spoilage and pathogenic micro-organisms associated with foods

Antimicrobial activity of seven commercial smoke preparations (four liquid and three solid) was studied. The minimum inhibitory concentration (MIC) was determined against a selection of food spoilage and pathogenic micro-organisms. The main smoke components were identified and quantified by gas chromatography/mass spectrometry. The most effective condensate was S2. All strains except Salmonella enteritidis were inhibited by S2 with an MIC <0·5–1·5%. Smoke extract L2 inhibited growth of Vibrio vulnificus, Yersinia enterocolitica, Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, L. inocua, Brochothrix thermosphacta and Lactococcus lactis ssp. lactis with an MIC of <0·2–0·8%. The condensate L3 inhibited effectively V. vulnificus, B. subtilis, L. innocua and Staph. aureus. L1, L4, S1 and S3 had no inhibitory effects at levels tested against most micro-organisms. Vibrio vulnificus was the most susceptible micro-organism to test compounds. The antimicrobial activity of smoke preparations was related to the concentration of phenols.     

Effect of various kanamycin dosage forms in experimental nonpenetrating infected injuries to the cornea]

Animals with experimental non-penetrating local wound of the cornea of the type of erosion infected with highly virulent strains of Staph. aureus were treated at various periods, i.e. in 15 minutes, 2, 6 and 9 hours with kanamycin solution (10000 gamma/ml) and kanamycin imbibized films. The therapeutic effect of the kanamycin solution on the non-penetrating infected wound of the cornea was higher when the treatment was started at earlier periods after infection with Staph. aureus. At later periods when the inflammatory process was highly developed the effect of the kanamycin solution on the wound was lower. As for the use of the kanamycin films the regularity was the reverse: they had a higher therapeutic effect in pronounced inflammation and highly developed infection of the eye.     

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.     

Investigation of functional and morphological changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Origanum compactum essential oil

Aims:  Evaluation of the cellular effects of Origanum compactum essential oil on Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213.
Methods and Results:  The damage induced by O. compactum essential oil on these two strains has been studied using different techniques: plate count, potassium leakage, flow cytometry (FC) and transmission electron microscopy (TEM). The results showed that oil treatment led to reduction of cells viability and dissipated potassium ion gradients. Flow cytometric analysis showed that oil treatment promoted the accumulation of bis-oxonol and the membrane-impermeable nucleic acid stain propidium iodide (PI), indicating the loss of membrane potential and permeability. The ability to reduce 5-cyano-2,3-ditolyl tetrazolium chloride was inhibited. Unlike in Ps. aeruginosa , membrane potential and membrane permeability in Staph. aureus cells were affected by oil concentration and contact time. Finally, TEM showed various structural effects. Mesosome-like structures were seen in oil-treated Staph. aureus cells whereas in Ps. aeruginosa, coagulated cytoplasmic material and liberation of membrane vesicles were observed, and intracellular material was seen in the surrounding environment. Both FC and TEM revealed that the effects in Ps. aeruginosa were greater than in Staph. aureus .
Conclusions:  Oregano essential oil induces membrane damage showed by the leakage of potassium and uptake of PI and bis-oxonol. Ultrastructural alterations and the loss of cell viability were observed.
Significance and Impact of the Study:  Understanding the mode of antibacterial effect of the oil studied is of a great interest in it further application as natural preservative in food or pharmaceutical industries.     

Prophylactic effect of a therapeutic vaccine against TB based on fragments of Mycobacterium tuberculosis

The prophylactic capacity of the RUTI® vaccine, based on fragmented cells of Mycobacterium tuberculosis, has been evaluated in respect to aerosol challenge with virulent bacilli. Subcutaneous vaccination significantly reduced viable bacterial counts in both lungs and spleens of C57Bl mice, when challenged 4 weeks after vaccination. RUTI® protected the spleen less than BCG. Following a 9 month vaccination-challenge interval, protection was observed for the lungs, but not for the spleen. Survival of infected guinea pigs was prolonged by vaccination given 5 weeks before challenge. Inoculations of RUTI® shortly after infection significantly reduced the viable bacterial counts in the lungs, when compared with infected control mice. Thus, vaccination by RUTI® has potential for both the prophylaxis and immunotherapy of tuberculosis.     

Comparative antibiotic sensitivity of different species of staphylococci isolated from humans]

A total of 206 strains of various staphylococcal species isolated from various sources were studied with respect to their sensitivity to 18 antibiotics. The number of strains poly-resistant to the antibiotics was almost the same among Staph. aureus and Staph. epidermidis, i. e. 54.8 and 51.3 per cent respectively. The coagulase-negative and mannitol-negative variants of Staph. aureus and Staph. epidermidis possessing high biological activity (10-14 properties) were resistant to more antibiotics as compared to the low active strains.     

Combination of hydrostatic pressure and lacticin 3147 causes increased killing of Staphylococcus and Listeria

The use of hydrostatic pressure and lacticin 3147 treatments were evaluated in milk and whey with a view to combining both treatments for improving the quality of minimally processed dairy foods. The system was evaluated using two foodborne pathogens: Staphylococcus aureus ATCC6538 and Listeria innocua DPC1770. Trials against Staph. aureus ATCC6538 were performed using concentrated lacticin 3147 prepared from culture supernatant. The results demonstrated a more than additive effect when both treatments were used in combination. For example, the combination of 250 MPa (2.2 log reduction) and lacticin 3147 (1 log reduction) resulted in more than 6 logs of kill. Similar results were obtained when a foodgrade powdered form of lacticin 3147 (developed from a spray dried fermentatation of reconstituted demineralized whey powder) was evaluated for the inactivation of L. innocua DPC1770. Furthermore, it was observed that treatment of lacticin 3147 preparations with pressures greater than 400 MPa yielded an increase in bacteriocin activity (equivalent to a doubling of activity). These results indicate that a combination of high pressure and lacticin 3147 may be suitable for improving the quality of minimally processed foods at lower hydrostatic pressure levels.     

Inhibition of Staphylococcus aureus by the commensal bacteria of human milk

AIMS: To study the bacterial diversity in expressed human milk with a focus on detecting bacteria with an antimicrobial activity against Staphylococcus aureus, known as a causative agent of maternal breast infections and neonatal infections. METHODS AND RESULTS: Random isolates (n = 509) were collected from breast milk samples (n = 40) of healthy lactating women, genotypically identified, and tested for antimicrobial activity against Staph. aureus. Commensal staphylococci (64%) and oral streptococci (30%), with Staph. epidermidis, Strep. salivarius, and Strep. mitis as the most frequent isolates, were the predominant bacterial species in breast milk. One-fifth of Staph. epidermidis and half of Strep. salivarius isolates suppressed growth of Staph. aureus. Enterococci (Ent. faecalis), isolated from 7.5% of samples, and lactic acid bacteria (LAB) (Lactobacillus rhamnosus, Lact. crispatus, Lactococcus lactis, Leuconoctoc mesenteroides), isolated from 12.5% of samples, were also effective against Staph. aureus. One L. lactis isolate was shown to produce nisin, a bacteriocin used in food industry to prevent bacterial pathogens and spoilage. CONCLUSIONS: Expressed breast milk contains commensal bacteria, which inhibit Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The strains inhibitory against the pathogen Staph. aureus have potential use as bacteriotherapeutic agents in preventing neonatal and maternal breast infections caused by this bacterium.     

Standardization of salt aggregation test for reproducible determination of cell-surface hydrophobicity with special reference to Staphylococcus species

The laboratory conditions for reproducible routine determination of staphylococcal cell-surface hydrophobicity by the salt aggregation test were standardized. Fresh bacterial suspensions standardized to 5 times 10⁹ cfu/ml gave the most reproducible results with both Staphylococcus aureus and coagulase-negative staphylococci. For relatively hydrophobic strains a 5-min reading time was necessary to detect bacterial aggregation in ammonium sulphate solutions ranging from 0.1 M to 1.5 M, pH 6.8. A × 10 hand lens facilitated reading aggregations. Overnight storage of bacterial suspensions at 20C reduced cell-surface hydrophobicity of all species, while storage at 4C reduced the hydrophobic nature of Staph. aureus strains. The hydrophobicity of coagulase-negative staphylococci rarely changed at 4C. A 10-fold dilution of fresh, standardized bacterial suspensions made it impossible to detect bacterial aggregation in ammonium sulphate solutions even with a hand lens. Under standardized conditions three types of staphylococcal cell aggregations were observed. The first looked like the slide agglutination for O antigens of Enterobacteriaceae, the second resembled H-agglutination, while the third had a filamentous appearance. These patterns indicated that more than one component might contribute to cell-surface hydrophobicity of both Staph. aureus and coagulase-negative staphylococci, or the same component might have different position on the cell surface.