Biological characteristics of spectinomycin-resistant strains of the tularemia pathogen

Sensitivity of 2 subspecies of the tularemia causative agent to spectinomycin, an aminoglycoside antibiotic, was studied in vitro. The MIC of the antibiotic with respect to strains 503/847 and Schu was 40 micrograms/ml and with respect to strain A-Cole 20 micrograms/ml. The frequency of spontaneous spectinomycin resistant mutants was low. The mutants grown on a medium containing spectinomycin in a concentration of 100 micrograms/ml were highly resistant to the antibiotic (at least 10000 micrograms/ml). By the main biological properties and virulence the spectinomycin resistant mutants did not differ from the initial strains.     

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.     

Sensitivity of beta-hemolytic streptococci to antibiotics]

Susceptibility of 64 beta-hemolytic streptococcal strains isolated from the patients with sore throat was studied by the method of serial dilutions in fluid nutrient medium (Konikov broth). Heterogenecity with respect to the sensitivity was investigated in 34 strains among separate populations of the microbes (10 to 15 in every strain). The MIC of benzylpenicillin, oxacillin and erythromycin ranged within 0.007--0.24 U/ml, 0.02--0.36 gamma/ml and 0.005--0.1 gamma/ml respectively. The MIC of benzylpenicillin with respect to separate populations most sensitive to it was 0.007--0.015 U/ml, while that with respect to the lease sensitive populations ranged from 0.015 to 0.24 U/ml. The respective values for oxacillin were 0.02--0.12 and 0.18--0.36 gamma/ml and those for erythromycin were 0.005--0.025 and 0.05--0.1 gamma/ml. Therefore, the beta-hemolytic streptococci isolated from the patients with sore throat were characterized by a rather high sensitivity to the antibiotics which was important precondition for their efficiency in treatment of the patients with the above disease.     

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.     

Characterization of a plasmid from the ruminal bacterium Selenomonas ruminantium

A 4.8-kilobase-pair plasmid was isolated from the ruminal bacterium selenomonas ruminantium HD4 by using a sodium carbonate-EDTA washing buffer to improve cell lysis (R.G. Dean, S.A. Martin, and C. Carver, Lett. Appl. Microbiol. 8:45-48, 1989). This plasmid, designated pSR1, appears to be quite stable. No evidence of plasmid DNA was detected in S. ruminantium D or GA192. All three strains were tested for antibiotic resistance, and all were kanamycin resistant (MIC, 25 to 50 micrograms/ml). Only strain D was tetracycline resistant (MIC, 25 micrograms/ml), and all strains were sensitive to ampicillin (MIC, 1 microgram/ml). pSR1 was cloned into pBR322, and a map of pSR1 was constructed by using HindIII, ClAI, BamHI, and PvuII. Although ClaI, BamHI, ScaI, and EcoRV digested recombined plasmid isolated from Escherichia coli, these restriction endonucleases were not effective in digesting plasmid isolated directly from S. ruminantium HD4.     

Characterization of a plasmid from the ruminal bacterium Selenomonas ruminantium.

A 4.8-kilobase-pair plasmid was isolated from the ruminal bacterium selenomonas ruminantium HD4 by using a sodium carbonate-EDTA washing buffer to improve cell lysis (R.G. Dean, S.A. Martin, and C. Carver, Lett. Appl. Microbiol. 8:45-48, 1989). This plasmid, designated pSR1, appears to be quite stable. No evidence of plasmid DNA was detected in S. ruminantium D or GA192. All three strains were tested for antibiotic resistance, and all were kanamycin resistant (MIC, 25 to 50 micrograms/ml). Only strain D was tetracycline resistant (MIC, 25 micrograms/ml), and all strains were sensitive to ampicillin (MIC, 1 microgram/ml). pSR1 was cloned into pBR322, and a map of pSR1 was constructed by using HindIII, ClAI, BamHI, and PvuII. Although ClaI, BamHI, ScaI, and EcoRV digested recombined plasmid isolated from Escherichia coli, these restriction endonucleases were not effective in digesting plasmid isolated directly from S. ruminantium HD4.     

Susceptibility of Neisseria meningitidis Strains from the Civilian Population to Sulfadiazine, Penicillin, and Rifampin

The minimal inhibitory concentration (MIC) values of sulfadiazine, penicillin, and rifampin for meningococcal strains isolated from civilians during 1970 were compared. The strains were isolated from various sources and geographical areas and represented several serogroups. The ranges of MIC values were as follows: 0.05 to 20 mg/100 ml for sulfadiazine, 0.01 to 0.4 mug/ml for penicillin, and 0.01 to 0.8 mug/ml for rifampin. There was no significant relationship between MIC values of sensitive or resistant sulfadiazine strains and the MIC values to the other two antimicrobial agents. Comparisons of sulfadiazine MIC values with inhibition zones around sulfathiazole discs showed excellent correlation, provided the strains were separated into sensitive and resistant groups on the basis of growth at 1 mg/100 ml. Regression curves for penicillin and rifampin sensitivity showed homologous sensitive populations with the strains studied.     

Involvement of multiple genetic determinants in high-level methicillin resistance in Staphylococcus aureus.

A methicillin-susceptible, novobiocin-resistant strain of Staphylococcus aureus (RN2677; methicillin MIC, 0.8 micrograms/ml) was transformed with DNA prepared from highly and homogeneously methicillin-resistant S. aureus strains (methicillin MIC, greater than or equal to 400 micrograms/ml) or from heterogeneous strains in which the majority of cells had a low level of resistance (methicillin MIC, 6.3 micrograms/ml). All methicillin-resistant transformants showed low and heterogeneous resistance (methicillin MIC, 3.1 micrograms/ml) irrespective of the resistance level of DNA donors. All transformants examined produced normal amounts of the low-affinity penicillin-binding protein (PBP) 2a, and methicillin resistance and the capacity to produce PBP 2a showed the same degree of genetic linkage to the novobiocin resistance marker with both homogeneous and heterogeneous DNA donors. Next, we isolated a methicillin-susceptible mutant from a highly and homogeneously resistant strain which had a Tn551 insertion near or within the PBP 2a gene and thus did not produce PBP 2a. With this mutant used as the recipient, genetic transformation of the methicillin resistance gene was repeated with DNA isolated either from highly and homogeneously resistant strains or from heterogeneous (low-resistance) strains. All transformants obtained expressed high and homogeneous resistance and produced PBP 2a irrespective of the resistance level of the DNA donors. Our findings suggest that (i) the methicillin resistance locus is identical to the structural gene for PBP 2a, (ii) although the ability to produce PBP 2a is essential for resistance, the MICs for the majority of cells are not related to the cellular concentration of PBP 2a, and (iii) high MICs and homogeneous expression of resistance require the products of other distinct genetic elements as well.     

Comparative evaluation of various bacterial growth inhibitors as selective agents for isolation of soil Actinomyces]

Tobramycin and dioxidine sensitivity of 57 strains belonging to 14 actinomycetes genera was studied. The cultures of Streptomyces were much more sensitive to tobramycin than the cultures of rare genera. The majority of the Streptomyces cultures showed a high resistance to dioxidine (MIC greater than or equal to 50 micrograms/ml). At the same time the majority of the cultures of rare genera were inhibited by low concentrations of dioxidine (no more than 50 micrograms/ml). For isolation of actinomycetes from soil samples, tobramycin, dioxidine, ceftriaxone and novobiocin were used. Tobramycin added in a concentration of 10 micrograms/ml to the Gauze agar organic medium No. 2 promoted a 2-fold increase in detection of actinomycetes of the rare genera as compared to the control. It was especially favourable for detection of cultures belonging to Micromonospora, Amycolatopsis, Streptosporangium and Nocardiopsis. Dioxidine in concentrations of 10 and 50 micrograms/ml inhibited the growth of the cultures belonging to rare genera. Ceftriaxone in the same concentrations inhibited the growth of the cultures of both Streptomyces and the rare genera. Novobiocin favoured detection of the cultures belonging to Amicolatopsis and Micromonospora. Therefore, among the tested compounds tobramycin and novobiocin appeared to be the most useful selective agents for isolation of actinomycetes of rare genera.     

Study of the activity of a new glycopeptide antibiotic eremomycin combined with tobramycin against Staphylococci in vitro

Eremomycin is an original natural antibiotic with glycopeptide structure isolated at the Institute of New Antibiotics, the USSR Academy of Medical Sciences. Activity of eremomycin alone or in combination with tobramycin was studied with using 25 clinical strains of staphylococci. 56 and 88 per cent of the strains were respectively resistant to gentamicin and kanamycin, two aminoglycoside antibiotics. All the staphylococcal strains were sensitive to eremomycin in concentrations of 0.12 to 1 microgram/ml. The MIC of tobramycin for 10 (40 per cent) sensitive strains ranged within 0.25-2 micrograms/ml. For 60 per cent of the strains the MIC was equal to or higher than 16 micrograms/ml. When eremomycin was used in combination with tobramycin the antibacterial effect with respect to 17 strains (68 per cent) increased. In 32 per cent of the strains the effect was synergistic and in 36 per cent of the strains it was additive. Indifference and antagonism were detected with respect to 7 (28 per cent) and 1 (4% per cent) strains respectively. No significant difference was shown in manifestation of the synergistic-additive nature of eremomycin and tobramycin interaction with respect to the tobramycin sensitive and resistant strains.     

The drug sensitivity of meningococci and the characteristics of its determination

The results obtained in the study of antibiotic and sulfamide sensitivity of 197 Neisseria meningitidis strains of groups A, B and C, isolated from the spinal fluid and blood of patients with meningococcal infection hospitalized in the 2nd Clinico-Infectious Hospital, Moscow, in 1984-1989 and studied with the use of the disc diffusion method and the method of serial dilutions of antibiotics in solid culture media, are presented. As revealed in this study, N. meningitidis strains retained their high sensitivity to penicillin and ampicillin (MIC50 = 0.016 and 0.032 micrograms/ml respectively). Sensitivity to tetracycline decreased (MIC50 = 0.5 micrograms/ml) and to rifampicin increased (MIC50 = 0.063 micrograms/ml). 48.5% of strains were resistant to streptomycin. In recent years the proportion of N. meningitidis, resistant to sulfanilamide preparations, significantly decreased and MIC50 was equal to 2.5 micrograms/ml in comparison with 5-10 micrograms/ml in the preceding period. The results of testing sensitivity to antibiotics by both methods coincided. Still the disc diffusion method can be used in epidemiological surveillance on meningococcal infection, while for more exact differentiation of N. meningitidis strains the use of the method of serial dilutions is necessary.     

Effects of chlorhexidine diacetate on Candida albicans, C. glabrata and Saccharomyces cerevisiae.

The effects of chlorhexidine diacetate (CHA) on Candida albicans, C. glabrata and wild-type and mannan, and permeability mutants of Saccharomyces cerevisiae have been studied. A CHA concentration of 10 micrograms/ml had little lethal activity against the Candida strains, but was more effective against S. cerevisiae. Concentrations of 100 and especially 1000 micrograms/ml brought about a much more rapid death of cells. 2-Mercaptoethanol enhanced the activity of CHA to some extent. Some of the mutant strains of S. cerevisiae were rather more sensitive than the wild-type strain. The age of cultures of C. albicans and C. glabrata influenced their response to CHA.     

Susceptibility of sixty-five non-oral clinical isolates of the Streptococcus milleri group to seven antimicrobial agents.

Antimicrobial susceptibilities of sixty-five non-oral Streptococcus milleri group clinical isolates to penicillin, gentamicin, lincomycin, ampicillin, chloramphenicol, tetracycline and erythromycin were determined by an agar dilution method. All strains were penicillin-sensitive (MIC < or = 0.031 microgram/ml) and the majority (64/65) were susceptible to erythromycin (MIC < or = 0.125 microgram/ml). Low-level resistance to gentamicin was observed, and the majority of strains possessed an MIC of 8 micrograms/ml. Lincomycin and ampicillin at 0.5 microgram/ml inhibited 52/65 and 61/65 strains, respectively. Of the isolates 92% were inhibited by chloramphenicol at < or = 2 micrograms/ml. Twenty-two S. milleri group strains (of which thirteen were vaginal isolates) were resistant to tetracycline (MIC > or = 8 micrograms/ml).     

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).     

The lipid A 1-phosphatase of Helicobacter pylori is required for resistance to the antimicrobial peptide polymyxin

Modification of the phosphate groups of lipid A with amine-containing substituents, such as phosphoethanolamine, reduces the overall net negative charge of gram-negative bacterial lipopolysaccharide, thereby lowering its affinity to cationic antimicrobial peptides. Modification of the 1 position of Helicobacter pylori lipid A is a two-step process involving the removal of the 1-phosphate group by a lipid A phosphatase, LpxEHP (Hp0021), followed by the addition of a phosphoethanolamine residue catalyzed by EptAHP (Hp0022). To demonstrate the importance of modifying the 1 position of H. pylori lipid A, we generated LpxEHP-deficient mutants in various H. pylori strains by insertion of a chloramphenicol resistance cassette into lpxEHP and examined the significance of LpxE with respect to cationic antimicrobial peptide resistance. Using both mass spectrometry analysis and an in vitro assay system, we showed that the loss of LpxEHP activity in various H. pylori strains resulted in the loss of modification of the 1 position of H. pylori lipid A, thus confirming the function of LpxEHP. Due to its unique lipid A structure, H. pylori is highly resistant to the antimicrobial peptide polymyxin (MIC > 250 microg/ml). However, disruption of lpxEHP in H. pylori results in a dramatic decrease in polymyxin resistance (MIC, 10 microg/ml). In conclusion, we have characterized the first gram-negative LpxE-deficient mutant and have shown the importance of modifying the 1 position of H. pylori lipid A for resistance to polymyxin.     

Relative sensitivities of environmental legionellae to selective isolation procedures

A survey of water samples to determine the efficacy of standard procedures for the isolation of environmental legionellae was conducted. Marked variations in intraspecies resistance to selective agents and treatments were observed, and in experiments with one of the isolates, the response was modified by culture conditions. Five selective procedures incorporating acid (pH 2.2) and heat (50 degrees C, 30 min) treatments, with and without plating on buffered charcoal-yeast extract agar supplemented with vancomycin (5 micrograms/ml), polymyxin B (60 U/ml), and cycloheximide (80 micrograms/ml), caused 5 to 99% decreases in viable counts of pure cultures in water suspensions. The differences in the responses of the cultures to the five treatments were statistically significant. Cells in retained samples of naturally contaminated water from which the original cultures had been isolated were significantly less sensitive than artificially grown isolates. The sensitivities of the laboratory-grown cells to the treatments were affected by the length of incubation on buffered charcoal-yeast extract agar. Whereas acid resistance increased after 24 h of incubation, resistance to the antibiotic mixture decreased.     

Relative sensitivities of environmental legionellae to selective isolation procedures.

A survey of water samples to determine the efficacy of standard procedures for the isolation of environmental legionellae was conducted. Marked variations in intraspecies resistance to selective agents and treatments were observed, and in experiments with one of the isolates, the response was modified by culture conditions. Five selective procedures incorporating acid (pH 2.2) and heat (50 degrees C, 30 min) treatments, with and without plating on buffered charcoal-yeast extract agar supplemented with vancomycin (5 micrograms/ml), polymyxin B (60 U/ml), and cycloheximide (80 micrograms/ml), caused 5 to 99% decreases in viable counts of pure cultures in water suspensions. The differences in the responses of the cultures to the five treatments were statistically significant. Cells in retained samples of naturally contaminated water from which the original cultures had been isolated were significantly less sensitive than artificially grown isolates. The sensitivities of the laboratory-grown cells to the treatments were affected by the length of incubation on buffered charcoal-yeast extract agar. Whereas acid resistance increased after 24 h of incubation, resistance to the antibiotic mixture decreased.     

Antibiotic Susceptibilities of Serratia marcescens and Enterobacter liquefaciens

Production of 5'-nucleotides by Serratia marcescens and Enterobacter liquefaciens correlates with deoxyribonuclease production, indicating the close relationship between these two organisms. To determine further relationships, susceptibilities of 279 strains of the tribe Klebsielleae were determined by the high-potency disc method, agar-dilution method, or both, by using 14 antibiotics. Ninety-seven per cent of S. marcescens (201 of 207 strains) and 100% of E. liquefaciens (17 strains) had minimum inhibitory concentration (MIC) of 100 mug/ml or greater with colistin and polymyxin B. With these two antibiotics, 93% of other Enterobacter species (28 strains) had MIC values of less than 1.6 mug/ml, and 100% of Klebsiella (27 strains) had MIC values less than 1.6 mug/ml. Consistent patterns were not noted with the other antibiotics tested, but the results with colistin and polymyxin B provide additional evidence of the close relationship of S. marcescens and E. liquefaciens.     

Induced resistance to kanamycin and tetracycline in Rhizobium japonicum

Spontaneous mutants Km-R and Tc-R of R. japonicum with various levels of resistance to kanamycin (0.8-20 mg/ml) and tetracycline (130-210 micrograms/ml) were isolated. No cross resistance in the mutants was observed. Plasmid R68.45 transferred to the wild strains resistance to 210 micrograms/ml of tetracycline and to 20 mg/ml of kanamycin. This plasmid did not practically increase the resistance to tetracycline in mutants Tc-R. At the same time it markedly increased the resistance to kanamycin in mutants Km-R.     

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.