Effect of subinhibitory levels of selected antibiotics on susceptibility of Staphylococcus aureus strains to phagocytosis and killing by rabbit granulocytes

The aim of the study was to assess the of chosen antibiotics in subinhibitory concentrations (sub-MIC) on the sensitive of Staphylococcus aureus cells to phagocytosis and killing by rabbit granulocytes. The following antibiotics were used: cloxacillin, cefadroxil, cefuroxim, cefotaxim, gentamicin, netilmicin, lincomicin, doxycycline and riphamicin. A total of 144 S. aureus strains with varied sensitivity to these antibiotics were selected for the study. The experiment used granulocytes isolated from rabbit blood and S. aureus strains incubated for 18 h in TSB broth containing antibiotics in the concentrations of 0.1 MIC, 0.2 MIC and 0.5 MIC, and in the antibiotics-free medium. Phagocytosis was assessed by the method of differential staining with acridine orange and crystal violet, allowing simultaneous determination of phagocytised and killed S. aureus cell counts. The findings revealed that the culture of S. aureus in the presence of all the antibiotics used in subinhibitory concentrations increased significantly the susceptibility of most S. aureus strains to phagocytosis and killing by granulocytes. The above effect usually occurred in the concentrations of 0.1 MIC (54.2%), more seldom in 0.2 MIC (13%) and 0.5 MIC (15% of strains). Each group of S. aureus contained some which showed no change in susceptibility following culture with the chemotherapeutic agents in subinhibitory concentrations (26.3%). Insensitive strains to the subinhibitory effects were equally common among susceptible (27%), intermediate (23%) and resistant (26%) strains of S. aureus to the antibiotics used. No statistically significant reduction was noted in phagocytosis or killing by rabbit granulocytes. No correlation was observed between the susceptibility to the subinhibitory effects of the antibiotics involved and their biochemical mechanisms.     

Sensitivity of the causative agent of tularemia to antibiotics

Sensitivity of the tularemia causative agent of different geographical races to antibiotics such as streptomycin, tetracycline, gentamicin, rifampicin (20 strains), ampicillin, polymyxin M, erythromycin, oleandomycin (361 strains) and lincomycin (294 strains) was studied. High sensitivity of the tularemi a microbe to streptomycin, tetracycline, rifampicin (MIC of 10 gamma/ml), gentamicin (MIC of 1 gamma/ml) and resistance to 50 gamma of ampicillin and 1000 gamma/ml of polymyxin M were found. Combined use of 50 gamma of ampicillin and 100 gamma/ml of polymyxin M added to the nutrient medium for growth inhibition of the foreign flora on isolation of the tularemia causative agent from the infected material including stable laboratory animal carcases was recommended. Marked differences in sensitivity of the strains of different geographical races to the macrolides and lincomycin were observed. The strains of the non-Arctic and Central Asiatic races were of low resistance to the above drugs (the MIC of erythromycin, oleandomycin and lincomycin were 10--50, 50--400 and 25--100 gamma/ml respectively. Within the holarctic race 40 per cent were low resistant and 60 per cent were highly resistant to these drugs. The above drugs should not be used for treatment of tularemia cases.     

Study of the sensitivity of the L-forms of group A Streptococcus to antibiotics in artificial nutrient media and in human cell cultures

Sensitivity of L-forms of group A streptococci to 5 antibiotics such as erythromycin, lincomycin, tetracycline, gentamicin and chloramphenicol was studied in an artificial nutrient medium and cell cultures i.e. human fibroblast diploid cells and transplantable human heart cells (Girardi). In vitro investigation of the antibiotic effect on the streptococcal L-forms revealed their sensitivity to erythromycin (MIC, 0.4 micrograms/ml), lincomycin (MIC, 0.08 microgram/ml) and tetracycline (MIC, 2 micrograms/ml). The streptococcal L-forms were slightly sensitive to gentamicin (MIC, 6 micrograms/ml) and chloramphenicol (MIC, 30 micrograms/ml). Complete inhibition of the growth of the L-forms in the Girardi cells on the 1st day of the experiment after the antibiotics administration in single doses was induced by lincomycin, 5 micrograms/ml, erythromycin, 10 micrograms/ml, and tetracycline, 100 micrograms/ml. In the diploid cells, the respective figures were 50, 100 and 200 micrograms/ml. Chloramphenicol and gentamicin had an inhibitory effect on the growth of the L-forms but produced no sanative effect.     

The effects of subminimal inhibitory concentrations of beta-lactam antibiotics against Clostridium perfringens.

The effects of subminimal inhibitory concentrations (sub-MIC) of four beta-lactam antibiotics [penicillin-G (PCG), ampicillin (AMP), cephaloridine (CER), cephalothin (CET)] were tested against Clostridium perfringens type A PB6K, after determining the minimum inhibitory concentrations (MIC) of 29 different Clostridium strains. The majority of the strains were sensitive to all beta-lactam antibiotics. Morphological changes, such as filamentous development and lysis, occurred at concentrations considerably lower than the MIC of CER and CET in C. perfringens. Clear cooperation of AMP and CER with rabbit polymorphonuclear leucocytes (PMNL) against C. perfringens was observed. The filamentous bacteria produced as a result of exposure to sub-MIC of each antibiotic, were phagocytosed easily. The ratios between the drug concentrations (microg/ml) at which the morphological changes began to occur, the minimum antibiotic concentrations (MAC), and the MIC values (microg/ml), were calculated. A large ratio indicated a wide range of effective concentrations below the MIC value for the antibiotics.     

The sensitivity to 12 antibiotics of 125 strains of Streptococcus group B isolated in a maternity home

Sensitivity of 125 strains of group B streptococci isolated from newborns, their mothers and personnel in a maternity home was studied with respect to 12 antibiotics: benzylpenicillin, ampicillin, methicillin, cephalotin, erythromycin, lincomycin, levomycetin (chloramphenicol), oxacillin, tetracycline, streptomycin, gentamicin and ristomycin. The method of serial dilutions in a solid medium was applied. All the strains were sensitive to ristomycin and erythromycin. The predominating number of the strains were sensitive to lincomycin, levomycetin and the beta-lactam antibiotics. Strains resistant or moderately resistant to benzylpenicillin, ampicillin, oxacillin, methicillin and cephalotin were detected. The majority of the strains were resistant to streptomycin, tetracycline and gentamicin. Multiple antibiotic resistance with 2-7 determinants was revealed in 11.2 per cent of the strains. The antibiotic sensitivity of the strains isolated from the newborns, their mothers and the personnel in the maternity home was on the whole similar or insignificantly differed.     

Antibiotic sensitivity of plague microbe strains from foreign countries]

The method of serial dilutions on the Hottinger agar was applied to comparative assay of antibiotic sensitivity in 50 strains of the plague microbe isolated abroad and in 5 strains isolated in the plague focus in the Central Caucasus. The antibiotics used in the assay were the following: streptomycin, gentamicin, doxycycline, monomycin, kanamycin, tetracycline, erythromycin, ristomycin, lincomycin and polymyxin M. Irrespective of the origin, all the isolates were resistant to erythromycin, lincomycin and polymyxin M. The levels of the sensitivity to the other antibiotics were different. The data serve as a ground for the statement that there is no tendency to development of antibiotic resistance in the plague microbe in patients treated with high doses of the antibiotics and mainly streptomycin. Along with streptomycin, such antibiotics as gentamicin, tetracycline, doxycycline and kanamycin are useful in the therapy of plague and require further investigation.     

Susceptibility to selected chemotherapeutics of Staphylococcus aureus strains resistant to methycyline isolated from clinical materials in the years 1991-1992 and 1997

The susceptibility to selected chemotherapeutic agents was determined in 100 strains of Staphylococcus aureus methicillin-resistant (MRSA) isolated from clinical materials in 1991-1992 (50 strains) and in 1997 (50 strains). Two methods were used for the determination: disc method and antibiotic dilution in agar. The minimal inhibitory concentration (MIC) was determined for vancomycin, teicoplanin, furazolidone, nitrofurantoin, ofloxacin, gentamicin, netilmicin and trimethoprim. The concentrations of the chemotherapeutics in the substrate ranged from 0.125 to 512 mg/l. The obtained results served for drawing of the following conclusions: all studied MRSA strains isolated in 1991-1992 and in 1997 were sensitive to glycopeptide antibiotics: vancomycin and teicoplanin, to nitrofurans: nitrofurantoin and furazolidone, and to fusidic acid. MRSA strains isolated in 1991-1992 were sensitive to ofloxacin, but in 1997 about 80% of the strains were resistant to that antibiotic, and this resistance was noted in S. aureus strains with homogeneous resistance to methicillin. Increasing frequency of resistance to mupirocin was found, in 1991-1992 4% of the strains were resistant, and in 1997 the resistance of MRSA to that antibiotic was found in 12%. No changes occurred in the sensitivity of staphylococci to trimethoprim/sulfamethoxazole (cotrimoxazole). About 94% of strains in 1991-1992 and 1997 were sensitive to that drug. The sensitivity to cotrimoxazole is connected with one of its components (trimethoprim), with 94% of MRSA strains sensitive to it.     

Sensitivity of the wound microflora of oncological patients to some new antibiotics]

Sensitivity of the microflora of the oncological patients' wounds to the new antibiotics, such as gentamicin, kanamycin, oxacillin, ampicillin and lincomycin was studied with the help of the disc method. The discs with the above antibiotics were prepared under laboratory conditions in accordance with the respective instructions in the WHO. Sensitivity of 429 bacterial cultures, including 98 cultures of pathogenic staphylocci, 45 cultures of Enterococci, 43 hemolytic streptococci, 143 cultures of Escherichia, 50 cultures of Ps. aeruginosa and 50 cultures of Proteus was determined. The studies showed that gentamicin was the most active antibiotic aganist all the microbial species isolated from the surgical and other wounds of oncological patients. It may be used in treatment of the infections caused by association of the microbes belonging to different species, as well as in treatment of purulent processes before elucidating their etiology, 16.7 per cent of the Enterococcal isolates were resistant to gentamicin. Monomycin, kanamycin, oxacillin, lincomycin and macrolide antibiotics at present are sufficiently active against pathogenic staphylococci and hemolytic streptococci.     

Desoxyribonuclease and lecithinase activity in antibiotic-resistant and -sensitive staphylococci]

Staphylococcus aureus, a laboratory strain 209-P and strain I isolated freshly from infected wounds, as well as lincomycin hydrochloride, ampicillin, oxacillin and methicillin manufactured in the USSR and cephaloridin manufactured by "PLIVA" in Yugoslavia were used. Various activity levels of desoxyribonuclease and lecitinase of the staphylococci depending on sensitivity or resistance of the test-microbe to the antibiotics were shown. The activity of the above microbial enzymes characterizing the pathogenic properties decreased with development of the antibiotic resistance, sometimes to complete inactivation of the enzymes synthesized by the staphylococci. In spite of closeness of their modes of action the semisynthetic penicillins had a differentiating effect on the above enzymes.     

Inhibition ofPseudomonas aeruginosa alginate expression by subinhibitory concentrations of antibiotics

The effects of subinhibitory concentrations (1/4, 1/8, 1/16 of the MIC) of quinolones (ciprofloxacin, enoxacin, nalidixic acid, norfloxacin, ofloxacin, pefloxacin), aminoglycosides (amikacin, gentamicin, netilmicin, streptomycin, tobramycin), β-lactams (aztreonam, ceftazidime, imipenem, ticarcilin) and macrolides (erythromycin, roxitromycin) on the excretion of alginate by aP. aeruginosa strain were studied. Both β-lactam and macrolide antibiotics were found ineffective at the concentrations tested, except erythromycin and imipenem at 1/4 MIC. Aminoglycosides at a concentration of 1/4 MIC reduced most effectively the excretion of alginate. Quinolones were also effective at this sub-MIC; 1/16 MIC was ineffective with all antibiotics or stimulated the production of alginate.     

Antibiotic sensitivity of staphylococci isolated from the surface of the skin of the breasts in pregnant women

Nine hundred and fourty coagulase-positive and coagulase-negative strains of staphylococci isolated from the skin surface of the mammary glands of 94 pregnant women were tested by the disc agar diffusion method for their sensitivity to five antibiotics. The highest number of the isolates were sensitive to erythromycin and lincomycin (87.7 and 89.7 per cent, respectively). The highest number of moderately resistant strains were detected with respect to methicillin. 19 out of 42 cultures of Staph. aureus were resistant to benzylpenicillin and 24 cultures were resistant to tetracycline. Among staphylococci 130 strains or 13.8 per cent were polyresistant.     

Results of 10 years of use of lincomycin (1966-1976) in the clinics of the N. N. Priorov Central Research Institute of Traumatology and Orthopedics]

During 10 years 1063 patients were treated with lincomycin used parentally or orally at the N. N. Priorov Central Research Institute of Traumatology and Orthopedy. The doses and the rate of its use depended on the state of the patient, its age and weight. Lincomycin was used for the treatment of patients with osteomyelitis or purulent wound infection, as well as for prophylaxis of suppuration. The drug was used for a long period of time under conditions of the same hospital, and it was shown that it remained up to the present days highly effective in therapy of infections and especially bone infections caused by staphylococci sensitive to it. The 10-year study of staphylococcal sensitivity to lincomycin revealed an insignificant increase in the development of resistance to it. The paper presents data on the importance of adequate surgical interventions in addition to the antibiotic therapy in cases with bone infections. A possibility of lincomycin combined use with other antibiotics and gentamicin or kanamycin in particular was shown. Complications, such as diarrhea and urticaria were registered in 11 patients.     

Antibiotic sensitivity of the microflora isolated in ENT diseases]

Sensitivity of beta-hemolytic streptococci of group A, Streptococcus viridans, Staphylococcus aureus, epidermal staphylococci, pneumococci, Proteus and Ps. aeruginosa isolated in 1975-1978 from patients with tonsillitis, otitis, sinusitis and other otorhinolaryngological diseases was studied with respect to 19 antibiotics. Data on comparison of the antibiotic sensitivity of the microflora isolated from the patients with otorhinolaryngological diseases in 1964-1974 and 1975-1978 are presented. It was shown that beta-hemolytic streptococci were highly sensitive to all the antibiotics tested except tetracycline. Among Streptococcus viridans the strains resistant to many antibiotics were more frequent than among beta-hemolytic streptococci. Most of the Staphylococcus aureus strains were sensitive to gentamycin, cephaloridin, oxacillin and resistant to the other antibiotics. The epidermal staphylococci were characterized by approximately the same antibiotic sensitivity as Staphylococcus aureus. Resistance of the predominating majority of the Pneumococcus strains to tetracycline was noted. Proteus and Ps. aeruginosa were resistant to all the antibiotics except aminoglycosides. The microflora isolated from the cases with otorhinolaryngological diseases in 1975-1978 were mainly characterized by lower antibiotic sensitivity than that isolated from the cases with the same diseases in 1964-1974. It is possible to suppose that the microorganisms isolated from the patients with otorhinolaringological diseases had no significant differences with respect to their antibiotic sensitivity from those isolated from the patients with other pathological processes.     

BioTimer Assay, a new method for counting Staphylococcus spp. in biofilm without sample manipulation applied to evaluate antibiotic susceptibility of biofilm

The medical device-related infections are frequently a consequence of Staphylococcus biofilm, a lifestyle enhancing bacterial resistance to antibiotics. Antibiotic susceptibility tests are usually performed on planktonic forms of clinical isolates. Some methods have been developed to perform antibiotic susceptibility tests on biofilm. However, none of them counts bacterial inoculum. As antibiotic susceptibility is related to bacterial inoculum, the test results could be mistaken. Here, a new method, BioTimer Assay (BTA), able to count bacteria in biofilm without any manipulation of samples, is presented. Moreover, the BTA method is applied to analyze antibiotic susceptibility of six Staphylococcus strains in biofilm and to determine the number of viable bacteria in the presence of sub-inhibitory doses of four different antibiotics. To validate BTA, the new method was compared to reference methods both for counting and antibiotic susceptibility tests. A high agreement between BTA and reference methods is found on planktonic forms. Therefore, BTA was employed to count bacteria in biofilm and to analyze biofilm antibiotic susceptibility. Results confirm the high resistance to antibiotics of Staphylococcus biofilm. Moreover, BTA counts the number of viable bacteria in the presence of sub-inhibitory doses of antibiotics. The results show that the number of viable bacteria depends on sub-inhibitory doses, age of biofilm and type of antibiotic. In particular, differently to gentamicin and ampicillin, sub-inhibitory doses of ofloxacin and azithromycin reduce the number of viable bacteria at lower extent in young than in old biofilm. In conclusion, BTA is a reliable, rapid, easy-to-perform, and versatile method, and it can be considered a useful tool to analyze antibiotic susceptibility of Staphylococcus spp. in biofilm.     

Failure of antibiotics gentamycin, tylosin, lincomycin and spectinomycin to eliminate Mycoplasma bovis in artificially infected frozen bovine semen

To study the effect of antibiotics upon Mycoplasma bovis in fresh bovine semen just before freezing, specimens of bovine semen were artificially infected with 1 of 9 different strains of M. bovis. Inocula of each strain were prepared to contain 10(5) to 10(6)/mL colony-forming units of M. bovis at 3 different stages of the growth phase. The infected semen was diluted with a Tris extender by a 3-step procedure using an antibiotic mixture of gentamicin, tylosin, lincomycin and spectinomycin (GTLS). This semen-antibiotic mixture was placed into French straws that were stored at -196 degrees C. The control semen specimens contained no antibiotics Mycoplasmas were counted after 8 d of storage in 3 decimal dilutions of the frozen semen. No evident effect was noticed upon the 9 tested strains of mycoplasmas in the semen frozen with the antibiotics, compared with that of the untreated control samples. It was further shown that this lack of effect was irrespective of the stage of the growth phase of the mycoplasmas. It was concluded that the antibiotic mixture (GTLS) in semen specimens is not capable of total elimination of mycoplasmas in frozen bovine semen.     

Sensitivity to antibiotics of pneumococci isolated from patients with chronic nonspecific pneumonia and bronchitis

Four hundred and twenty two pneumococcal strains isolated from 300 patients with chronic nonspecific pneumonia and bronchitis were studied with respect to their sensitivity to 18 antibiotics within a period from 1982 to 1985. It was shown with the method of serial dilutions on solid media that 91.7, 87.8, 85 and 81 per cent of the isolates were sensitive to benzylpenicillin, ampicillin, lincomycin and cefuroxime, respectively. A significant percentage of the pneumococcal strains had decreased sensitivity to benzylpenicillin (MIC close to the therapeutic concentration). On this basis it was recommended to use lower concentrations of benzylpenicillin (less than 0.25 units/ml) in assay of sensitivity in clinical strains of Pneumococcus.     

Production of toxic shock exotoxin by Staphylococcus aureus strains resistant and sensitive to antibiotics

Three hundred and ninety two strains of S. aureus isolated from bacteria carriers and patients with staphylococcal infections in different regions of the Soviet Union were investigated. 55.9 per cent of the isolates were able to produce exotoxin of toxic shock. No regular relation between resistance to definite antibiotics (tetracycline, chloramphenicol, benzylpenicillin, streptomycin, lincomycin, erythromycin, oleandomycin, gentamicin and methicillin) and the polyresistance range on the one hand and the ability to produce toxic shock exotoxin on the other hand was revealed.     

Impact of antibiotics on conjugational resistance gene transfer in Staphylococcus aureus in sewage

The growing rate of microbial pathogens becoming resistant to standard antibiotics is an important threat to public health. In order to assess the role of antibiotics in the environment on the spread of resistance factors, the impact of subinhibitory concentrations of antibiotics in sewage on gene transfer was investigated using conjugative gentamicin resistance (aacA-aphD) plasmids of Staphylococcus aureus. Furthermore, the concentration of antibiotics in hospital sewage was measured by high-performance liquid chromatography (HPLC)-electrospray tandem mass spectrometry. Several antibiotics were found to be present in sewage, e.g. ciprofloxacin up to 0.051 mgl(-1) and erythromycin up to 0.027 mgl(-1). Resistance plasmid transfer occurred both on solidified (dewatered) sewage and in liquid sewage in a bioreactor with a frequency of 1.1x10(-5)-5.0x10(-8). However, low-level concentrations of antibiotics measured in sewage are below concentrations that can increase plasmid transfer frequencies of gentamicin resistance plasmids of staphylococci.     

Bacterial multiresistance to antibiotics. Study of types of bacterial resistance to antibiotics in the environment and in clinical material of a resuscitation ward

Types of resistance were studied in clinical material and in the environment a resuscitation ward in the period from September 1973 to February 1974. Identical strains with the same types of resistance were isolated in both groups. They were verified by qualitative and quantitative tests of sensitivity to selected antibacterial substances. A high degeee of resistance to streptomycin, chloramphenicol, tetracycline and ampicillin was found in all gram-negative bacilli. High effectivity of colimycin, gentamicin and co-trimoxazole was confirmed in vitro. The strains of Staphylococcus aureus were fully sensitive only to oxacillin, lincomycin, spiramycin, vancomycin, gentamicin and co-trimoxazole. The strains of Streptococcus faecalis were found sensitive only to ampicillin and vancomycin.     

Long-term antibiotic susceptibility pattern of strains of Neisseria gonorrhoeae isolated in Czechoslovakia in the years 1957-1982

Over the past 25 years a total of 7492 strains of Neisseria gonorrhoeae have been isolated in Czechoslovakia, mainly in Prague (64%). All these strains have been tested for susceptibility to the following antibiotics: penicillin G, ampicillin, tetracycline, spectinomycin, erythromycin, doxycycline, kanamycin, rifampin, chloramphenicol, gentamicin, cephalothin, cephaloridine, lincomycin and clindamycin. In addition, seven derivatives of newer antibiotics of penicillin and cephalosporin series were tested in 1981. The study showed that in 1957 the MIC of 0.03 units of penicillin per ml was effective against 95% of strains, but in 1981 only 37% of isolates were sensitive to this concentration. The first gonococcal strains with the MIC value of 4.0 units/ml to penicillin were detected in 1981. This tendency towards decreased gonococcal susceptibility to benzylpenicillin is alarming. Over the last eight years there have been described sporadic isolations of strains relatively resistant to tetracycline (MIC = 8.0 mg/l). The susceptibility to spectinomycin has been tested in over 4000 gonococcal strains, since 1967. The test showed that this antibiotic remained highly effective against the gonococcal infection with over 95% of gonococci with the MIC value of 16.0 mg/l. No fully spectinomycin resistant strains have been found. Penicillin G as well as spectinomycin and cefotaxim are still considered the antibiotics of the first choice in the treatment of gonorrhoea. The alternative antibiotics may include cefuroxim, chloramphenicol and, in cases of sensitive strains, tetracyclines.