Effect of antibiotic and vaccine therapies on the succinate dehydrogenase and phosphatase activity of liver cells of mice infected with Staphylococcus

Changes in the activity of succinate dehydrogenase (SDH), total and acid phosphatase (TP and AP) were studied in treatment of laboratory animals with rifampicin, lincomycin and with inactivated staphylococcal vaccine used alone or in combinations. It was shown that immunization of the animals with inactivated staphylococcal vaccine under conditions of experimental staphylococcal infection promoted stimulation of the enzyme activity. Rifampicin and lincomycin used for the treatment of such animals lowered the activity of the enzymes. The suppressing effect of the antibiotics increased with an increase in the period of their use. It should be noted that the inhibitory effect of rifampicin on the activity of SDH, TP and AP was less pronounced than that of lincomycin. The combined use of the vaccine and antibiotics for the treatment of the animals promoted an increase in the enzyme activity as compared to the use of the antibiotics alone. Sometimes the activity of SDH, TP and AP reached the control levels in such animals or the levels observed in the animals treated with the vaccine alone. Stimulation of the enzyme activity was more pronounced when the vaccine was used in combination with rifampicin.     

Changes in the serum acid phosphatase activity of guinea pigs poisoned with C1 perfringens type A toxin and a mixture of toxin and a broth culture filtrate

It was shown in experiments on guinea pigs that the injection of C1. perfringens type A toxoid induced an increase in acid phosphatase activity of animal blood serum. The action of the toxoid increased under the effect of C1. butyricum cultural filtrate, which gave rise to an earlier enhancement of the specific activity of the enzyme as compared to the injection of the toxoid alone. Increased activity of acid phosphatase may play a pathogenetic role in cases of anaerobic infection caused by association of C1. perfringens and C1. butyricum.     

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.     

Molecular characterization and antimicrobial resistance of Clostridium perfringens isolates of different origins from Costa Rica

Clostridium perfringens, a Gram positive, spore-forming anaerobe, is widely distributed in nature. Based upon their production of four major toxins alpha, beta, epsilon and iota, C. perfringens is classified into five toxinotypes (A-E). Some strains produce an enterotoxin (CPE), encoded by the cpe gene, which causes diarrhea in humans and some animals. C. perfringens strains that had been previously isolated and been kept at -80 degrees C were analyzed for the presence of toxin genes and for antimicrobial resistance: 20 from soils, 20 from animal, 20 from human origin and 21 from food non related to outbreaks. According to PCR results, all strains were classified as C. perfringens type A, since only alpha toxin gene was detected, while cpe was detected in two strains (2.5%) isolated from food, as it has been described in other world regions. Antibiotic resistance to at least one antibiotic was detected in 44% of the strains, 41% was resistant to clindamycin, 25% to chloramphenicol, 22% to penicillin and 20% to metronidazole. Soils strains showed the highest resistance percentages to almost all antibiotics. Multiresistance (to three or more antibiotic groups) was detected in the strains from soil (40%), human origin (30%), food (14%) and animal origin (5%). The high resistance rates found may be explained by the widespread use of antimicrobials as growth promoters in plants and animals; also these resistant strains may act as reservoir of resistance genes that may be transferred between bacteria in different environments.     

Effect of antibiotic therapy and vaccine therapy on the phagocytic reaction in mice infected with Staphylococcus

The time course of changes in the activity, intensity and completeness of phagocytosis with leukocytes of the peritoneal exudate was studied on mice with experimental staphylococcal infection treated with rifampicin, lincomycin and inactivated staphylococcal vaccine used alone or in combination. It was shown that immunization of the animals with inactivated staphylococcal vaccine promoted stimulation of the phagocytic defense. Rifampicin and lincomycin applied therapeutically induced a decrease in the activity, intensity and completeness of phagocytosis. It should be noted that rifampicin had a less pronounced inhibitory effect than lincomycin. The combined use of vaccine and antibiotics with therapeutic purposes promoted an increase in phagocytosis as compared to the use of the antibiotics alone. The combined therapy sometimes resulted in completeness of phagocytosis making it reach the control values (the 10th and 15th days, rifampicin and vaccine). It should be noted that a more pronounced stimulation of the activity, intensity and completeness of the phagocytosis was observed with the use of the combination of rifampicin and the vaccine.     

Characterization of Bacillus cereus strains isolated from drugs and evaluation of their toxins

The microbial contamination of 68 samples of topical and 324 samples of oral medicaments has been studied. The most common group of contaminants was members of the genus Bacillus (34.4%). Because of the pathogenic significance of B. cereus, 39 strains were characterized by morphology and biochemical properties. All except three showed most of the characteristics of the type strain. They were highly resistant to lincomycin, polymyxin B and penicillin G-cephalosporin and were susceptible to streptomycin, erythromycin and chloramphenicol. Enterotoxin, phospholipase C and haemolysin production were also studied: 33 strains gave a positive vascular permeability reaction, four of them causing necrosis, and 24 showed positive mouse lethal tests. All the strains had phospholipase activity. The majority also exhibited differing degrees of haemolysis. Permeability factor was related to mouse lethality and haemolytic activity. Phospholipase C was not related to any of the above activities.     

Characterization of Bacillus cereus strains isolated from drugs and evaluation of their toxins

The microbial contamination of 68 samples of topical and 324 samples of oral medicaments has been studied. The most common group of contaminants was members of the genus Bacillus (34.4%). Because of the pathogenic significance of B. cereus , 39 strains were characterized by morphology and biochemical properties. All except three showed most of the characteristics of the type strain. They were highly resistant to lincomycin, polymyxin B and penicillin G-cephalosporin and were susceptible to streptomycin, erythromycin and chloramphenicol. Enterotoxin, phospholipase C and haemolysin production were also studied: 33 strains gave a positive vascular permeability reaction, four of them causing necrosis, and 24 showed positive mouse lethal tests. All the strains had phospholipase activity. The majority also exhibited differing degrees of haemolysis. Permeability factor was related to mouse lethality and haemolytic activity. Phospholipase C was not related to any of the above activities.     

Large scale isolation of maternally inherited lincomycin resistance mutations, in diploid Nicotiana plumbaginifolia protoplast cultures

Summary Lincomycin-resistant clones were isolated in diploid protoplast cultures of Nicotiana plumbaginifolia. Selection of the resistant clones was based on the ability of resistant calli to green in the presence of the antibiotic (1,000 mg l^-1). Sensitive colonies formed white calli under the same conditions. In the absence of mutagenic treatment the frequency of the resistant clones was 1.0×10^-4. This frequency could be increased up to 5.8×10^-4 and 7.2×10^-4 by treatment with 0.1 mM and 0.3 mM N-ethyl-N-nitrosourea (NEU), respectively.Regenerated plants of 56 clones were tested for lincomycin resistance. Regenerates from all but seven clones were resistant to lincomycin, as demonstrated by leaf assay. The lincomycin-resistant regenerates tested were also resistant to clindamycin (a lincomycin derivative), but sensitive to streptomycin.Regenerated plants in 17 clones were fully fertile and inherited lincomycin resistance maternally. Segregation for lincomycin resistance was observed in the seed progeny of five clones, which indicated maintenance of mixed cytoplasmic determinants after plant regeneration. Seed transmission of lincomycin resistance was confirmed in an additional 17 clones but the mode of inheritance (maternal or Mendelian) was not determined because of pollen sterility or reduced seed germination ability. These defects first appeared when the higher concentration of NEU was used. Various pigment deficiencies were also observed in a few clones.     

The first strain of Clostridium perfringens isolated from an avian source has an alpha-toxin with divergent structural and kinetic properties

Clostridium perfringens alpha-toxin is a 370-residue, zinc-dependent, phospholipase C that is the key virulence determinant in gas gangrene. It is also implicated in the pathogenesis of sudden death syndrome in young animals and necrotic enteritis in chickens. Previously characterized alpha-toxins from different strains of C. perfringens are almost identical in sequence and biochemical properties. We describe the cloning, nucleotide sequencing, expression, characterization, and crystal structure of alpha-toxin from an avian strain, SWan C. perfringens (SWCP), which has a large degree of sequence variation and altered substrate specificity compared to these strains. The structure of alpha-toxin from strain CER89L43 has been previously reported in open (active site accessible to substrate) and closed (active site obscured by loop movements) conformations. The SWCP structure is in an open-form conformation, with three zinc ions in the active site. This is the first example of an open form of alpha-toxin crystallizing without the addition of divalent cations to the crystallization buffer, indicating that the protein can retain three zinc ions bound in the active site. The topology of the calcium binding site formed by residues 269, 271, 336, and 337, which is essential for membrane binding, is significantly altered in comparison with both the open and closed alpha-toxin structures. We are able to relate these structural changes to the different substrate specificity and membrane binding properties of this divergent alpha-toxin. This will provide essential information when developing an effective vaccine that will protect against C. perfringens infection in a wide range of domestic livestock.     

Experimental and clinical study test of capreomycin]

Data on the experimental and clinical study of capreomycin in the treatment of tuberculosis are presented. It was shown that capreomycin had low activity with respect to the sensitive strain of Mycobacterium tuberculosis H37 Rv in vitro and the respective infection caused by it in mice. The activity of capreomycin in vitro with respect to streptomycin resistant strains was the same as that with respect to the sensitive strains, while in vivo it increased 3 times. Capreomycin showed a tendency to decreasing its activity with respect to strains highely resistant to canamycin only in vitro. The effect of capreomycin on tuberculosis infection caused by strains resistant to different concentrations of canamycin was the same as that on tuberculosis infection caused by sensitive strains. Cross resistance between florimycin (viomycin) and capreomycin was shown. Clinical trails of capreomycin revealed its moderate therapeutic efficiency, relatively low toxicity and an allergenizing effect on the host. Transient ventibulopatia without pronounced signs of ototoxic action was observed. The nephrotoxic effect was moderate and transient. It was observed predominantly at the peak of the allergic reactions to the antitubercle drugs. The data obtained during the study allow recommendation of capreomycin use in clinics as reserve drug when the causative agent is not resistant to florimycin. The drug should be used under regular control of the blood picture, electrolyte metabolism, state of the kidneys, auditory and vestibular apparatus.     

Sensitivity of Mycoplasma hominis cultures isolated from clinical material to certain antibiotics]

Sensitivity to II antibiotics of 80 strains of M. hominis isolated from patients with various inflammatory processes of the urogenital tract was studied by the method of suppressing the metabolic activity. Inhibition of the arginine metabolism of mycoplasma was used as a test for determination of the growth suppression. All the strains tested were highly sensitive to tetracycline and lincomycin. Kanamycin and neomycin were less active against M. hominis. All the strains tested were resistant to erythromycin, oleandomycin, ristomycin, novobiocin and streptomycin. The inhibitory effect of tetracycline and lincomycin on M. hominis decreased by the 5th day.     

酪酸梭菌与婴儿型双歧杆菌对产气荚膜梭菌试管内生物拮抗作用的研究

用酪酸权菌（Clostridium butyricum）和婴儿型双歧杆菌（Bifidobacterium Infantis）对产气荚膜梭菌（Clostriduim perfringens）进行试管内的生物拮抗试验。将酪酸梭菌、婴儿型双歧杆菌及酪酸梭菌和婴儿型双歧杆菌分别对产气荚膜酸菌以一定的比例等量混合接种于GAM液体培养基中进行厌氧培养。实验证明酪酸梭菌和婴儿型双歧能明显抑制产气荚膜梭菌的生长,并且比各自单独培养时显示了较强的生物拮抗作用。     

Biochemical differentiation between enterotoxigenic heat-sensitive and heat-resistant Clostridium perfringens strains

Some biochemical characteristics of 37 enterotoxigenic Clostridium perfringens strains isolated from human feces, ground beef, and soil samples by heat-selection methods and of two NCTC strains were studied. Two different biochemical patterns closely related to the heat resistance of the strains were found. The strains placed into group 1 were trehalose, inositol, and sorbitol negative and synthesized heat-resistant spores, while those placed into group 2 were trehalose and inositol positive and synthesized heat-sensitive spores. Sorbitol fermentation was variable among the strains of this last group. The strains of group 1 were more cellobiose, melibiose, and salicin fermentative than those of group 2. Only the strains placed into group 2 synthesized toxins of sufficient levels for typing. In spite of having been isolated by mild heat treatment of the specimens, two strains showed the same biochemical and toxigenic characteristics of the strains of group 1. The heating of these two strains did not modify their characteristics. We conclude that enterotoxigenic C. perfringens strains showing the two different toxigenic and biochemical patterns are present in the human gut, ground beef, and, probably, in soil. These strains may be differentiated on the basis of their capacity to produce acid from trehalose, inositol, and sorbitol, heat resistance of the spores and grade of toxigenicity. The heat-selection methods used for isolation of C. perfringens strains from different sources exerted a selection of strains from one or another group, but had no influence on their toxigenic and biochemical properties.     

Use of the peroxidase method of immunoenzyme analysis for detecting Clostridium perfringens cells

The possibility of using the peroxidase method of the enzyme immunoassay for the detection of C. perfringens cells in the culture fluid of strains, as well as in affected tissues in cases of anaerobic gas gangrene infection, has been shown. This method is highly specific, convenient and takes but 1.5 hrs.     

Antimicrobial Susceptibility of Clostridium difficile from Different Sources

A total of 79 Clostridium difficile strains from healthy young and elderly adults, elderly patients without gastrointestinal disease, elderly patients receiving antibiotics without gastrointestinal complications, and elderly patients with antibiotic-associated diarrhea or pseudomembranous colitis were tested for their susceptibilities to 24 antimicrobial agents. All of the 79 strains were inhibited by low concentrations of rifampicin, metronidazole, fusidic acid, vancomycin, ampicillin, and penicillin G. The strains were highly resistant to aminoglycosides, trimethoprim, sulfamethoxazole, nalidixic acid, and cycloserine and often resistant to neomycin, cefoxitin, and cefalexin. Wide variations in the susceptibility of C. difficile strains to erythromycin, clindamycin, lincomycin, chloramphenicol, and tetracycline were found. Strains resistant to erythromycin, clindamycin, and lincomycin were more frequently found among strains isolated from elderly adults than those isolated from young adults, with particularly high frequency among strains isolated from elderly patients receiving antibiotics. None of the 23 strains isolated from healthy young adults was resistant to chloramphenicol. All of the 14 strains resistant to erythromycin, clindamycin, lincomycin, and chloramphenicol were sensitive to tetracycline and all of the 15 strains resistant to erythromycin, clindamycin, lincomycin, and tetracycline were sensitive to chloramphenicol. Only one out of 19 tetracycline-resistant strains was highly toxigenic, whereas 42 (70%) of 60 sensitive strains were highly toxigenic.     

Experimental criteria for interpretation of bacterial sensitivity to dioxidine determined by diffusion from the disks

Antibacterial activity of dioxidine against aerobic and facultative anaerobic organisms under conditions of anaerobiosis i. e. conditions really observed for example in abscess cavities or necrotic tissues is 30 to 100 times as high as that under aerobic conditions. There is a relationship between sensitivity of bacteria to dioxidine under aerobic and anaerobic conditions which is expressed by the regression equation. Therefore, comparison of the MICs determined under anaerobic conditions with the growth inhibition zones formed by disks with the drug under aerobic conditions is possible. The MIC of dioxidine was determined under anaerobic conditions for 179 clinical strains of aerobic and facultative anaerobic bacteria and the growth inhibition zones of the same bacteria under aerobic conditions were evaluated with the use of disks containing 100, 75, 50, 25, 20, and 15 micrograms of the drug. The border line. MIC differentiating between resistant and sensitive strains was chosen to be equal to 4 micrograms/ml. Differentiation of the strains into sensitive and resistant ones by the values of the growth inhibition zones was performed with the method of error minimization described by C. Metzler and R. De Haan in 1974. Disks containing 25 micrograms of the drug allowed one to differentiate the strains under aerobic conditions into sensitive and resistant ones: the growth inhibition zones greater than 11 mm corresponded to the sensitive strains (the MIC smaller than 4 micrograms/ml) and the growth inhibition zones smaller than 11 mm corresponded to the resistant strains (the MIC greater than 4 micrograms/ml).     

Prevalence and characterization of Clostridium perfringens from spices in Argentina

Spices can present high microbial counts and Clostridium perfringens, Bacillus cereus, Salmonella and Shigella, among others have been isolated from spices. C. perfringens is an important pathogen agent causing, among other diseases, enteritis in humans caused by C. perfringens enterotoxin (CPE) which causes human food poisoning and enterotoxemia in domestic animals. The aims of the present work were (i) to establish the hygienic sanitary quality of some spices in San Luis, Argentina; (ii) to determine the presence of C. perfringens in these spices by means of the most probable number (MPN) and count on plate methods; (iii) to characterize the enterotoxigenic strains of C. perfringens by PCR and immunological methods such as reverse passive latex agglutination (RPLA) and (iv) to type by PCR C. perfringens strains isolated. A total of 115 samples of spices, 67 of which were purchased in local retail stores and 48 domestically collected were analysed. Total aerobe counts on tryptone glucose yeast extract agar medium of the 115 samples were between <10 and 10(6) CFU/g. The colifecal counts using Mac Conkey broth of the 115 samples were <4-10(3)CFU/g, with 28 samples (24.34%) exceeding the limit established by the Spanish Alimentary Code (10 CFU/g) while 2 samples (1.73%) had a sulfite reducing anaerobe load above standard limits. A total of 14 C. perfringens strains (12.17%) were isolated and characterized from 115 samples by the standard biochemical tests. Four of which (28.60%) turned out to be enterotoxigenic by PCR and RPLA. In order to type C. perfringens strains based on their main toxins, the 14 strains were analysed by PCR. All strains belonged to type A. All RPLA positive strains were cpe(+) by PCR. The percentage of enterotoxigenic strains was more elevated that those reported in other studies for this type of sample. These results indicate that sanitary conditions in different production stages of species must be improved to reduce health hazards. The high percentage (24.34%) of samples with colifecal values above standard limits is an indication of deficient sanitary conditions. These results suggest the need to provide legislation on the sanitary and hygienic quality of spices in our country.     

Susceptibility of Clostridium perfringens to C-C fatty acids

AIMS: To determine susceptibility of Clostridium perfringens strains CCM 4435(T) and CNCTC 5459 to C(2)-C(18) fatty acids, and evaluate influence of pH in cultures grown on glucose. Straw particles were added to cultures to simulate the presence of solid phase of the digestive tract milieu. METHODS AND RESULTS: Antimicrobial activity of fatty acids was expressed as a concentration at which only 50% of the initial glucose was utilized. Lauric acid showed the highest antimicrobial activity, followed by myristic, capric, oleic and caprylic acid. Only strain CNCTC 5459 was susceptible to linoleic acid. Neither caproic acid and acids with a shorter carbon chain nor palmitic and stearic acid influenced substrate utilization. The antimicrobial activity of myristic, oleic and linoleic acid decreased when clostridia were grown in the presence of straw particles. In cultures of both strains treated with capric and lauric acid at pH 5.0-5.3, the number of viable cells was <10(2) ml(-1). Only lauric acid reduced number of viable cells of both strains below 10(2) ml(-1) at pH > 6. Transmission electron microscopy revealed separation of inner and outer membranes and cytoplasma disorganization in cells treated with lauric acid. CONCLUSIONS: Lauric acid had the highest activity towards C. perfringens among fatty acid tested. Its activity was not influenced by the presence of solid particles and did not cease at pH > 6. SIGNIFICANCE AND IMPACT OF THE STUDY: Lauric acid might be a means for control of clostridial infections in farm animals.     

Antimicrobial susceptibility of anaerobic bacteria in Bulgaria

ObjectivesThe antimicrobial susceptibility of anaerobic bacteria isolated from clinical specimens in the referent for Bulgaria anaerobic laboratory was studied in a period of 25 years/1983–2007/.MethodsNCCLS – recommended agar dilution methods were used. β-lactamase activity was determined with nitrocefin discs.ResultsThe 29 antimicrobial agents included in the study were divided according to their in vitro activity against the anaerobic isolates into 4 main groups for guiding empirical treatment: 1st group of metronidazole, chloramphenicol, meropenem, imipenem and combinations of β-lactam antibiotics with sulbactam – with high activity and drugs of choice for treatment; 2nd group – clindamycin, cefoxitin, carbenicillin/and azlocillin, piperacillin/ – with a good activity and low percent of resistant strains; 3rd group – of tetracycline and erythromycin with higher percent of resistant strains including the new macrolides as josamycin, clarithromycin, roxithromycin and azithromycin; 4th group – penicillins/ampicillin, amoxicillin, penicillin/and cephalosporins/cefamandole, cefazolin, cefotaxime and cefoperazone/ – not suitable for treatment of infections including Bacteroides fragilis group strains, with a very high percent of resistant strains, probably due to β-lactamase activity in most of the strains.ConclusionA continued updating and a follow-up in the changes of antibiotic susceptibility are necessary in every country as resistance patterns vary not only between geographical regions but also even among medical centers and hospitals which may be connected with differences in antibiotic usage in man and animals.