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.     

In vitro inhibitory effect of hen egg white lysozyme on Clostridium perfringens type A associated with broiler necrotic enteritis and its alpha-toxin production

AIMS: Clostridium perfringens type A causes both clinical and subclinical forms of necrotic enteritis in domestic avian species. In this study the inhibitory effect of hen egg white lysozyme on the vegetative form of Cl. perfringens type A and the production of alpha-toxin in vitro was investigated. METHODS AND RESULTS: A micro-broth dilution assay was used to evaluate the minimal inhibitory concentrations (MIC) of lysozyme against three clinical isolates of Cl. perfringens type A in 96-well microtitre plates. The MIC of lysozyme against Cl. perfringens isolates was found to be 156 microg ml(-1). Scanning electron micrographs of the cells treated with 100 microg ml(-1) of lysozyme revealed extensive cell wall damage. A quantitative sandwich ELISA for alpha-toxin produced by Cl. perfringens was developed based on a commercial ELISA kit allowing only qualitative detection. Addition of 50 microg ml(-1) of lysozyme did not inhibit the growth of Cl. perfringens but significantly inhibited the toxin production. CONCLUSIONS: Lysozyme inhibited the growth of Cl. perfringens type A at 156 microg ml(-1). At sublethal levels, lysozyme was able to inhibit the alpha-toxin production. SIGNIFICANCE AND IMPACT OF STUDY: Inhibition of Cl. perfringens type A and its alpha-toxin production by hen egg white lysozyme had never previously been reported. By inhibiting this avian pathogen and its toxin production, lysozyme showed potential for use in the treatment and prevention of necrotic enteritis and other Cl. perfringens type A related animal diseases.     

In vitro effect of ampicillin/sulbactam on Acinetobacter species

The effects of subinhibitory concentrations (sub-MIC) of ampicillin/sulbactam (AMP/sulbactam), on the surface hydrophobicity and the lipase activity of ten Acinetobacter strains, were examined. The alterations in the activities studied were strain and drug concentration dependent. Most of the strains treated showed a decrease in surface hydrophobicity to a different extent. The hydrophobic character of three strains exposed to 1/4 or 1/8 of the MIC of the antibiotic was changed to a hydrophilic state. The majority of Acinetobacter strains after treatment with antibiotic possessed increased lipolytic activity. AMP/sulbactam even at sub-MIC may interfere with possible virulence factors of Acinetobacter strains in vitro.     

Antifungal effects of hydrolysable tannins and related compounds on dermatophytes, mould fungi and yeasts

A series of hydrolysable tannins and related compounds was evaluated for antifungal activities against filamentous fungi (Epidermophyton floccosum; Microsporum canis; Microsporum gypseum; Trichophyton mentagrophytes; Trichophyton rubrum; Trichophyton tonsurans; Trichophyton terrestre; Penicillium italicum; Aspergillus fumigatus; Mucor racemosus; Rhizopus nigricans) and opportunistic yeasts (Candida albicans; Candida glabrata; Candidata krusei; Cryptococcus neoformans), using the agar dilution method. While all samples had no activity against the filamentous fungi in concentrations of 1.1-5.9 microM (1000 microg/ml), the phenolic compounds displayed significant potencies against all the opportunistic yeasts tested but C. albicans, with minimum inhibitory concentrations ranging from 0.02 to 0.1 microM (16-125 microg/ml). Although the presence of galloyl groups in flavonoids did not necessarily produce activity, this structural element, an HHDP moiety or its oxidatively modified entity proved to be an important structural feature of hydrolysable tannins. Comparison of dilution methods provided strong evidence of dependence of MIC values on the test method. Employing the microdilution broth method, the ellagitannin corilagin (MIC 0.8 nM) was found to be similarly potentially active as amphotericin B (MIC 0.5 nM) and sertaconazole (MIC 0.9 nM) against Candida glabrata strains. The order of effectiveness observed being 64- and 4-8-fold increased for corilagin and the reference compounds respectively, when compared with that of the agar dilution test.     

In vitro susceptibility of Clostridium perfringens isolated from farm animals to growth-enhancing antibiotics

Minimal inhibitory concentration (MIC) determinations were carried out with seven growth-enhancing antibiotics against 95 Clostridium perfringens field isolates obtained during 1991 and 1992 from poultry, pigs and calves. All were resistant to 64 μg ml⁻¹ of the bambermycin antibiotic, flavomycin (flavophospholipol) and susceptible to avoparcin (MIC₉₀ 0.25 μg ml⁻¹), avilamycin (MIC₉₀ 0.5 μg ml⁻¹) and salinomycin (MIC₉₀≤ 0.12 μg ml⁻¹). Acquired resistance against bacitracin was detected in some isolates from poultry and bovines and resistance to tylosin and virginiamycin in some strains from all species investigated. Overall, the prevalence of resistance was comparable to the low levels recorded in 1979 in Cl. perfringens isolates from the same animal host species.     

In vitro anti-Neisseria gonorrhoeae activity of Terminalia macroptera leaves

We used the agar dilution method to evaluate the antibacterial effect of Terminalia macroptera leaf (Tml) extract against nine reference and clinical Neisseria gonorrhoeae strains, including penicillin- and tetracycline-resistant and -susceptible strains. Tml possesses anti-N. gonorrhoeae activity against all of the strains and the minimum inhibitory concentrations (MIC) were between 100 and 200 microg ml(-1). We then used a liquid-liquid partition method to divide the Tml extract into five fractions and determined the anti-N. gonorrhoeae activity of each of the fractions. All of the fractions showed antibacterial activity. The most active one was identified as the diethyl ether fraction and had MIC values of between 25 and 50 microg ml(-1) against all of the strains.     

Intermedilysin release by Streptococcus intermedius: effects of various antibacterial drugs at sub-MIC levels

Intermedilysin is a cytolytic toxin produced by Streptococcus intermedius, a pathogen of humans. In vitro studies showed that exposure of S. intermedius to sub-minimum inhibitory concentration (MIC) levels (1/2 MIC) of protein-inhibiting antibiotics and nucleic acid-inhibiting antibiotics decreased intermedilysin release by S. intermedius. The most potent antibiotic was clindamycin. On the other hand, exposure to cell wall-inhibiting antibiotics generally showed insignificant changes in intermedilysin release at sub-MIC concentrations. Investigations into possible mechanisms underlying this sub-MIC effect with clindamycin showed that there was selective decrease in biosynthesis and release of toxin after exposure to 1/2 MIC condition. However, no significant differences in the mRNA levels of the intermedilysin gene were observed.     

Isolation of 1,4-naphthalenedione, an antibacterial principle from the leaves of Holoptelea integrifolia and its activity against beta-lactam resistant Staphylococcus aureus

Antimicrobials derived from plants have been receiving increasing attention in recent years. Antimicrobial activities of a number of phytochemicals have been reported. Many present day antibiotics are ineffective against several pathogenic organisms. About 90% of Staphylococcus aureus isolates from clinical specimens is reported to have resistance against beta-lactam antibiotics. In the present study, the effect of hexane, diethyl ether, acetone and water extracts of leaves of a medicinal plant Holoptelea integrifolia has been tested against beta-lactam resistant strain of S. aureus in presence of antibiotics such as ampicillin, amoxicillin, cefotaxime and ceftriaxone. The diethyl ether extract has shown the maximum antibacterial activity and the active principle is found to be 1,4-naphthalenedione which is characterized by GC-MS and FTIR spectroscopy. The minimum inhibitory concentration (MIC) of the compound is found to be 4 mg/ml. Structural similarity of this compound with a functional group of a beta-lactamase-resistant antibiotic indicates that 1,4-naphthlenedione may be acting as an inhibitor to beta-lactamase.     

In vitro efficacy of ciprofloxacin alone and in combination with amoxycillin against Salmonella typhi isolates

Combined effect of ciprofloxacin (Ci) and amoxycillin (Ax) has been studied in vitro against 12 clinical isolates of S. typhi that showed Ci minimum inhibitory concentration (MIC) of > or =1 microg/ml. By agar dilution method, MIC values of Ax were 10-16 microg/ml for 11 isolates and 0.5 microg/ml for the remaining one isolate. The isolates, when treated with Ci and Ax in combination, showed fractional inhibitory concentration (FIC) of 0.004-0.256 microg/ml for Ci. FIC of Ax ranged from 6-10 microg/ml, except for a single isolate that showed Ax FIC of 0.25 microg/ml. Thus Ci was more efficacious in combination with Ax against S. typhi than Ci alone. The antibiotic combination exhibited an additive effect for all the isolates showing FIC index 0.504-0.832.     

Comparison of the effects in vitro of tea tree oil and plaunotol on methicillin-susceptible and methicillin-resistant strains of Staphylococcus aureus

The effects in vitro of tea tree oil (TTO) and plaunotol were examined by monitoring the growth of a standard strain of Staphylococcus aureus FDA 209P and of fourteen methicillin-susceptible strains of S. aureus (MSSA), together with twenty methicillin-resistant strains (MRSA). The minimum inhibitory concentrations (MIC) and the doses for 50% inhibition of growth (ID50) were determined by the micro-broth dilution (MD) method, and the broth dilution with shaking (BDS) method, respectively. The MIC of plaunotol for 50 and 90% of the MSSA and MRSA were assessed by the MD method, as 16 microg/ml and > or = 1,024 microg/ml, respectively. No antibacterial effects of TTO on MSSA and MRSA were detected by the MD method. The growth-inhibitory effects of TTO on S. aureus by the BDS method were examined, and it appeared that TTO was effective over a lower range of concentrations than previously reported. It seems that TTO is very effective in vitro against MSSA and MRSA at high concentrations but less effective below 40 microg/ml of TTO.     

The anti-staphylococcal activity of Angelica dahurica (Bai Zhi)

Bioassay-guided fractionation of a hexane extract prepared from the roots of the Chinese drug Angelica dahurica (Bai Zhi) led to the isolation of the polyacetylenic natural product falcarindiol (1). The absolute stereochemistry of this compound was confirmed by careful 1H NMR analysis of its (R)- and (S)-Mosher ester derivatives as the 3(R), 8(S) isomer. Activity was tracked using a Mycobacterium fortuitum screening assay and the purified product was evaluated against multidrug-resistant and methicillin-resistant strains of Staphylococcus aureus (MRSA). The minimum inhibitory concentrations (MIC) of this metabolite ranged from 8 to 32 microg/ml highlighting the potential of the acetylene natural product class as antibiotic-lead compounds. These MIC values compare favourably with some of the newest agents in development for the treatment of MRSA infection and indicate that further evaluation of the antibiotic activity of acetylenes is warranted.     

Antibacterial activity evaluation of microcin J25 against diarrheagenic Escherichia coli

The inhibitory activities of known microcins were evaluated against some diarrheagenic Escherichia coli strains. Some antibacterial properties of microcin J25, the most active one, were studied. A rapid two-step purification was performed. The MIC and the minimum bactericidal concentration of J25 against E. coli O157:H7 were 1 and 100 microg ml(-1), respectively. A 10(4)-CFU ml(-1) contamination by this strain was destroyed in milk and meat extract by 6.25 microg of J25 ml(-1) and in half-diluted egg yolk by 50 microg of J25 ml(-1).     

Synergistic effects of the combination of galangin with gentamicin against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The antimicrobial killing activity toward methicillin-resistant Staphylococcus aureus (MRSA) has been a serious emerging global issue. New effective antimicrobials and/or new approaches to settle this issue are urgently needed. The oriental herb, Alpinia officinarum, has been used in Korea for several hundreds of years to treat various infectious diseases. As it is well known, one of the active constituents of Alpinia officinarum is galangin. Against the 17 strains, the minimum inhibitory concentrations (MICs) of galangin (GAL) were in the range of 62.5 ~ 125 microg/ml, and the MICs of gentamicin (GEN) ranged from 1.9 microg/ml to 2,000 microg/ml. The fractional inhibitory concentrations (FICs) of GAL, in combination with GEN, against 3 test strains were 0.4, 3.9, and 250 microg/ml, and were all 15.62 microg/ml in GEN. The FIC index showed marked synergism in the value range of 0.19 to 0.25. By determining time-kill curves, also confirmed the low synergism of the GAL and GEN combination against 4 h, 8 h, 12 h, and 24 h cultured MRSA. The time-kill study results indicated a low synergistic effect against 3 test strains. Thus, the mixture of GAL and GEN could lead to the development of new combination antibiotics against MRSA infection.     

In vitro acitivity of ribostamycin against Prototheca sp.

The in vitro activity of ribostamycin against algae of the genus Prototheca was evaluated by minimum inhibitory concentration (MIC) studies on solid media. Concentrations of 4 mcg/ml were required to inhibit 100% of the P. zopfii strains; 16 mcg/ml inhibited 100% of the P. stagnora strains and 95% of the P. wickerhamii strains. These values are inferior to plasma concentrations obtained after injection of ribostamycin. It is likely that this antibiotic could be effective in the treatment of protothecosis in man.     

Kakadumycins,novel antibiotics from Streptomyces sp NRRL 30566, an endophyte of Grevillea pteridifolia

An endophytic streptomycete (NRRL 30566) is described and partially characterized from a fern-leaved grevillea (Grevillea pteridifolia) tree growing in the Northern Territory of Australia. This endophytic streptomycete produces, in culture, novel antibiotics - the kakadumycins. Methods are outlined for the production and chemical characterization of kakadumycin A and related compounds. This antibiotic is structurally related to a quinoxaline antibiotic, echinomycin. Each contains, by virtue of their amino acid compositions, alanine, serine and an unknown amino acid. Other biological, spectral and chromatographic differences between these two compounds occur and are given. Kakadumycin A has wide spectrum antibiotic activity, especially against Gram-positive bacteria, and it generally displays better bioactivity than echinomycin. For instance, against Bacillus anthracis strains, kakadumycin A has minimum inhibitory concentrations of 0.2-0.3 microg x ml(-1) in contrast to echinomycin at 1.0-1.2 microg x ml(-1). Both echinomycin and kakadumycin A have impressive activity against the malarial parasite Plasmodium falciparum with LD(50)s in the range of 7-10 ng x ml(-1). In macromolecular synthesis assays both kakadumycin A and echinomycin have similar effects on the inhibition of RNA synthesis. It appears that the endophytic Streptomyces sp. offer some promise for the discovery of novel antibiotics with pharmacological potential.     

Development of an accelerated method of determining the antibiotic sensitivity of Cl. perfringens type A]

An express method for determination of antibiotic sensitivity in the strains of Cl. perfringens of type A using Soviet dry nutrient media and antibiotics is proposed. The criteria for estimation of the level of the antibiotic sensitivity of the causative agent of gas gangrene in short periods on the basis of comparison of the data of the antibiotic agar diffusion procedure and the antibiotic MIC were worked out. Twelve antibiotics and 45 collection strains of Cl. perfringens of type A were used in the experiment. The antibiotic agar diffusion method with the use of the nutrient media, microbial load and cultivation conditions developed by the authors is recommended for tentative determination of the antibiotic sensitivity in Cl. perfringens of type A for 4 hours. The use of the agar diffusion method and determination of the antibiotic MIC provided complete estimation of tha antibiotic sensitivity of Cl. perfringens of type A within not more than 24 hours.     

A simple classification method for residual antibiotics using E. coli cells transformed by the calcium chloride method and drug resistance plasmid DNA

Using three different plasmid DNA codings for kanamycin (KM), chloramphenicol (CP), and ampicillin- (AMP) and tetracycline- (TC) resistance, four different competent Escherichia coli strains were transformed by the calcium chloride method to produce KM-, CP- and AMP- and TC-resistant strains. Evaluation of minimum inhibitory concentrations (MIC) of 22 antibiotics, showed KM-resistant E. coli to be cross resistant only to fradiomycin (FRM); CP-resistant E. coli, especially HB101 and JM109 strains, exhibited cross-resistance only to thiamphenicol (TP). On the other hand, AMP- and TC-resistant E. coli showed cross resistance to several penicillins, tetracyclines and erythromycin. E. coli ATCC-27166, the strain most sensitive to all drugs in this experiment, was employed for disc diffusion experiments and from the pattern of appearance of the inhibition zone, eight major antibiotics were divided into three groups depending on their activity against containing each of the three plasmids. Only gentamicin (GM) activity was not affected by any of the drug resistant strains. Assay techniques utilizing three resistant strains may be the technique for screening foods for antibiotic residues in the future.     

Isolation and characterization of Streptomyces sp KH-614 producing anti-VRE (vancomycin-resistant enterococci) antibiotics

The actinomycete strain KH-614 was antagonistic to vancomycin-resistant enterococci (VRE). Based on the diaminopimelic acid (DAP) type, morphological and physiological characteristics examined by scanning electron microscopy (SEM), KH-614 was confirmed as belonging to the genus Streptomyces. Based on the 16S rDNA nucleotide sequences, Streptomyces sp. KH-614 was found to have a relationship with Streptomyces lydicus. The production of antibiotic from this strain was most favorable when cultured in glucose, polypeptone, yeast extract (PY) medium for 6 days at 27 degrees C. The antibiotic was identified as a cyclo(L-leucyl-L-prolyl) by comparing it with the reported spectral data including MS and NMR. Cyclo(leu-pro) was found to be active against twelve VRE strains, including E. faecium (vanA, vanB), and E. faecalis (vanA, vanB), that had been isolated over a period three years (1998-2000). Cyclo(leu-pro) was especially effective against VRE strains such as E. faecalis (K-99-34), E. faecalis (K-00-184), E. faecalis (K-00-221), and the MIC values were 12.5 microg/ml. Moreover, cyclo(leu-pro) was effective against three leukemic cell lines at concentrations below 100 microg/ml. At 100 mg/ml cyclo(leu-pro), K562, HL60, and U937 leukemic cell lines showed growth inhibition of 95, 91, and 93%, respectively. In a normal cell line, MDBK, cyclo(leu-pro) exerted 24% growth inhibition at a concentration of 100 microg/ml, and showed no inhibitory activity at concentrations below 10 microg/ml. These results indicate that cyclo(leu-pro) is a potential anti-leukemic and anti-VRE agent.     

Antimicrobial activity of mupirocin, daptomycin, linezolid, quinupristin/dalfopristin and tigecycline against vancomycin-resistant enterococci (VRE) from clinical isolates in Korea (1998 and 2005)

It is a hot clinical issue whether newly approved antimicrobial agents such as daptomycin, linezolid, quinupristin/dalfopristin (synercid) and tigecycline are active enough to be used for infections caused by vancomycin resistant bacteria. We performed susceptibility tests for mupirocin, which is in widespread clinical use in Korea, and four new antimicrobials, daptomycin, linezolid, quinupristin/dalfopristin and tigecycline, against vancomycin-resistant Enterococcus faecalis and Enterococcus faecium isolated from Korean patients in 1998 and 2005 to evaluate and compare the in vitro activity of these antimicrobials. Among these agents, quinupristin/dalfopristin, which is rarely used in hospitals in Korea, showed relatively high resistance to several vancomycin-resistant enterococci (VRE) isolated in 2005. Likewise, daptomycin, linezolid and tigecycline have not yet been in clinical use in Korea. However, our results showed that most of the 2005 VRE isolates were already resistant to linezolid and daptomycin (highest minimum inhibitory concentration (MIC) value >100 microg/ml). Compared with the other four antimicrobial agents tested in this study, tigecycline generally showed the greatest activity against VRE. However, four strains of 2005 isolates exhibited resistance against tigecycline (MIC >12.5 microg/ml). Almost all VRE were resistant to mupirocin, whereas all E. faecium isolated in 1998 were inhibited at concentrations between 0.8 to approximately 1.6 microg/ml. In conclusion, resistances to these new antimicrobial agents were exhibited in most of VRE strains even though these new antibiotics have been rarely used in Korean hospitals.     

Antibiotic exposure as a risk factor for emergence of resistance: the influence of concentration

Evolution of antibiotic resistance (AR) is increasingly perceived as a major clinical problem. The use of bactericidal antibiotics may protect against this, to some extent, by eradication of the pathogen, but the borders between cidal and inhibitory activity in the patient are often blurred. In addition, there are clinical reasons why eradication of the pathogen may not always be desirable. Antibiotic dosing schedules are currently driven by the perception that T > MIC and AUIC are the main predictors of outcome for time-dependent and concentration-dependent antibiotics, respectively. In the context of protecting against development of resistance in the pathogen however, peak antibiotic concentration and the concept of mutant prevention concentrations may be more important. The role of post-antibiotic and sub-MIC effects is more conjectural. Considerations of mechanisms of resistance and their relationship to antibiotic dosing schedules will also be highlighted. Lastly, the relevance of all this to the development of resistance in the normal bacterial flora will be discussed.