Streptomycin-susceptible Strains Research Articles

Increased transmission of Mycobacterium tuberculosis Beijing genotype strains associated with resistance to streptomycin: a population-based study.

BackgroundStudies have shown that the Mycobacterium tuberculosis Beijing genotype is an emerging pathogen that is frequently associated with drug resistance. This suggests that drug resistant Beijing strains have a relatively high transmission fitness compared to other drug-resistant strains.Methods and FindingsWe studied the relative transmission fitness of the Beijing genotype in relation to anti-tuberculosis drug resistance in a population-based study of smear-positive tuberculosis patients prospectively recruited and studied over a 4-year period in rural Vietnam. Transmission fitness was analyzed by clustering of cases on basis of three DNA typing methods. Of 2531 included patients, 2207 (87%) were eligible for analysis of whom 936 (42%) were in a DNA fingerprint cluster. The clustering rate varied by genotype with 292/786 (37%) for the Beijing genotype, 527/802 (67%) for the East-African Indian (EAI) genotype, and 117/619 (19%) for other genotypes. Clustering was associated with the EAI compared to the Beijing genotype (adjusted odds ratio (ORadj) 3.4: 95% CI 2.8–4.4). Patients infected with streptomycin-resistant strains were less frequently clustered than patients infected with streptomycin-susceptible strains when these were of the EAI genotype (ORadj 0.6, 95% CI 0.4–0.9), while this pattern was reversed for strains of the Beijing genotype (ORadj 1.3, 95% CI 1.0–1.8, p for difference 0.002). The strong association between Beijing and MDR-TB (ORadj 7.2; 95% CI 4.2–12.3) existed only if streptomycin resistance was present.ConclusionsBeijing genotype strains showed less overall transmissibility than EAI strains, but when comparisons were made within genotypes, Beijing strains showed increased transmission fitness when streptomycin-resistant, while the reverse was observed for EAI strains. The association between MDR-TB and Beijing genotype in this population was strongly dependent on resistance to streptomycin. Streptomycin resistance may provide Beijing strains with a fitness advantage over other genotypes and predispose to multidrug resistance in patients infected with Beijing strains.

Streptomycin Resistance and Lineage-Specific Polymorphisms in Mycobacterium tuberculosis gidB Gene

Mutations related to streptomycin resistance in the rpsL and rrs genes are well known and can explain about 70% of this phenotypic resistance. Recently, the gidB gene was found to be associated with low-level streptomycin resistance in Mycobacterium tuberculosis. Mutations in gidB have been reported with high frequency, and this gene appears to be very polymorphic, with frameshift and point mutations occurring in streptomycin-susceptible and streptomycin-resistant strains. In this study, mutations in gidB appeared in 27% of streptomycin-resistant strains that contained no mutations in the rpsL or rrs genes, and they were associated with low-level streptomycin resistance. However, the association of certain mutations in gidB with streptomycin resistance needs to be further investigated, as we also found mutations in gidB in streptomycin-susceptible strains. This occurred only when the strain was resistant to rifampin and isoniazid. Two specific mutations appeared very frequently in this and other studies of streptomycin-susceptible and -resistant strains; these mutations were not considered related to streptomycin resistance, but as a polymorphism. We stratified the strains according to the different phylogenetic lineages and showed that the gidB(16) polymorphism (16G allele) was exclusively present in the Latin American-Mediterranean (LAM) genotype, while the gidB(92) polymorphism (92C allele) was associated with the Beijing lineage in another population. In the sample studied, the two characterized single-nucleotide polymorphisms could distinguish LAM and Beijing lineages from the other lineages.

Epidemiologic study of Taylorella equigenitalis strains by field inversion gel electrophoresis of genomic restriction endonuclease fragments

Contagious equine metritis (CEM), a sexually transmitted bacterial disease, was first described in thoroughbred horses. It also occurs in nonthoroughbred horses, in which it produces isolated, apparently unrelated outbreaks. Thirty-two strains of Taylorella equigenitalis, the causative agent of CEM, from all over the world were characterized by field inversion gel electrophoresis of fragments of genomic DNA obtained by digestion with low-cleavage-frequency restriction enzymes. This resulted in a division into five clearly distinct groups. Strains from thoroughbred horses from all continents belonged to one group. Strains from nonthoroughbred horses from various countries were different from strains from thoroughbred horses; four groups could be determined. Two groups contained both streptomycin-resistant and streptomycin-susceptible strains. The data indicate that CEM in nonthoroughbreds did not originate from the thoroughbred population; also, the reverse was not demonstrated. Thus, extensive international transportation directives regarding the testing of nonthoroughbred horses for CEM may need reconsideration.

Synergistic effect of ampicillin or vancomycin in combination with decreasing concentrations of streptomycin against enterococci.

The extent of synergistic killing and duration of the post-antibiotic effect (PAE) was evaluated in streptomycin-susceptible strains of Streptococcus faecalis and Streptococcus faecium with 20 mcg/ml of ampicillin or 10 mcg/ml of vancomycin in combination with decreasing concentrations of streptomycin (20, 10, 5, 2, 1, 0.5 mcg/ml). The bactericidal activity or duration in recovery period declined progressively in a linear pattern with decreasing concentrations of streptomycin. One log10 or greater reduction in viable counts was seen with streptomycin concentrations of 2 mcg/ml or higher with both ampicillin and vancomycin. A synergistic increase in recovery period (PAE greater than or equal to 0.5 h) of ampicillin or vancomycin was observed with concentrations of streptomycin in excess of 2 mcg/ml and 1 mcg/ml, respectively. These results suggest that lower dosage of streptomycin in the therapy of enterococcal infections will probably result in a higher relapse rate.

Comparison of the post-antibiotic effect of Streptococcus faecalis and Streptococcus faecium with ampicillin alone or combined with streptomycin: studies on a novel type of antimicrobial interaction.

Determination of post-antibiotic effect (PAE) and time-kill studies were made with twelve strains of Streptococcus faecalis and nine strains of Streptococcus faecium, comparing the effect of ampicillin alone with a combination of ampicillin and streptomycin at achievable serum concentrations. Bactericidal synergism (greater than or equal to one log10 decrease in viable counts) and prolongation in PAE (greater than or equal to 0.5 h) were demonstrated in all streptomycin-susceptible strains (Minimum inhibitory Concentration less than 2000 mcg/ml), whereas only one of five highly streptomycin-resistant strains exhibited a synergistic effect. A significant correlation between the magnitude of increased killing and the increase in recovery period by the combinations of ampicillin and streptomycin was demonstrated.

In vitro and in vivo studies of streptomycin-resistant, penicillin-susceptible streptococci from patients with infective endocarditis.

The combination of penicillin and streptomycin did not act synergistically in vitro against three streptomycin-resistant strains (MIC, greater than or equal to 1,000 micrograms of streptomycin/ml) of penicillin-susceptible streptococci. Using a model of experimental infective endocarditis, we infected rabbits with a control streptomycin-susceptible strain, with an intermediately streptomycin-resistant strain (MIC, 1,000 micrograms/ml), and with a highly streptomycin-resistant strain (MIC, greater than 32,000 micrograms/ml). Treating animals with a combination of procaine penicillin and streptomycin was more effective (P less than .01) than treating them with procaine penicillin alone only for those animals infected with the control streptomycin-susceptible strain. Treatment with procaine penicillin plus gentamicin was more effective (P less than .01) than treatment with procaine penicillin alone for all three treatment groups and was more effective (P less than .01) than treatment with procaine penicillin and streptomycin for those animals infected with an intermediately or highly streptomycin-resistant strain of streptococci.

Roles of ribosomal binding, membrane potential, and electron transport in bacterial uptake of streptomycin and gentamicin.

The effects of a set of conditions on aminoglycoside uptake were determined. Membrane vesicles either with a membrane potential (delta psi) of -125 mV (adequate to drive lysine uptake) or with succinate, lactate, or phenazine methosulfate did not accumulate gentamicin unless components of protein synthesis were included. Ribosomally resistant (rpsL) Escherichia coli cells demonstrated energy-dependent phase II uptake similar to that of a streptomycin-susceptible strain of E. coli when treated with 100 micrograms of puromycin per ml. Puromycin (100 micrograms/ml) also increased the uptake of the cationic compounds polyamine and arginine. These studies support a role of protein synthesis in aminoglycoside uptake and in the development of energy-dependent phase II. delta psi of cells did not increase either at the initiation of or during energy-dependent phase II, showing that energy-dependent phase II is not due to an elevation of delta psi. In a Bacillus subtilis system, significant streptomycin uptake requires a threshold value of delta psi which varies depending upon the concentration of streptomycin used. At 25 micrograms/ml, the uptake of streptomycin reached maximal levels after exceeding the threshold value, whereas at 100 micrograms/ml there was a gradual increase of the uptake to the maximal after the threshold value was exceeded. Several studies supported the view that electron transport has a specific role other than its requirement to produce the cellular delta psi. The uptake of gentamicin was stimulated to a greater extent by phenazine methosulfate-ascorbate than by the ionophore nigericin in strains of E. coli, although nigericin stimulated delta psi to a greater degree. Cells with 25% of the normal quinone concentration had delta psi values identical to cells with the normal quinone concentration, but the quinone-deficient cells had a significantly lower rate of gentamicin uptake. KCN prevented gentamicin uptake but did not prevent the development of delta psi. The effects of ubiquinone depletion in an E. coli strain were more evident on gentamicin uptake than on ATP-driven glutamine transport or proton motive force-driven proline transport, consistent with a specific requirement for quinones in aminoglycoside uptake. A detailed explanation of the mechanism of accumulation of streptomycin and gentamicin and a proposed mechanism for killing bacterial cells by these agents have been provided.

Anti-streptococcal activity of Sch 29482--a novel penem antibiotic

Sch 29482 (SCH), a new broad-spectrum penem antibiotic, inhibited viridans and β -haemolytic streptococci at concentrations of ≤ 13−0.5 mg/l. It killed group A streptococci, Streptococcus mulans , and Str. salivarius at concentrations of ≤ 0.13 mg/l. Its bactericidal effect varied with strains among other viridans streptococci tested. SCH, 16 mg/l, and gentamicin, 4mg/l, were synergistic against most of the streptomycin-susceptible strains of Str. faecalis tested but against only one streptomycin-resistant strain.

Minimal concentrations of aminoglycoside that can synergize with penicillin in enterococcal endocarditis.

Minimal concentrations of aminoglycoside that could produce a synergistic effect with penicillin were investigated in broth cultures containing 10(8) enterococci per ml, in vitro in vegetations infected with ca. 10(8) enterococci per g, and in vivo in an experimental model of enterococcal endocarditis. Penicillin G plus gentamicin (1.5 or 0.75 microgram/ml) sterilized a broth culture of a streptomycin-resistant strain (E1) at 48 h. In contrast, penicillin G plus gentamicin (1.5 or 0.75 microgram/ml) sterilized only 2 of 15 in vitro vegetations at 5 days. Similarly, doses of gentamicin that resulted in peak serum levels of 1.5 microgram/ml failed after 10 days of therapy with penicillin G plus gentamicin to sterilize in vivo vegetations infected with E1, and doses of gentamicin that resulted in peak serum levels of about 8 micrograms/ml sterilized four of six vegetations. Similar results were obtained with a streptomycin-susceptible strain. These studies indicated that the rate of bactericidal activity in broth cultures is greater than the bacteriological response in infected vegetations and that aminoglycoside concentrations that appear efficacious on the basis of synergy studies in broth cultures may not be satisfactory clinically.

Antimicrobial therapy of experimental enterococcal endocarditis.

The successful therapy of enterococcal endocarditis requires prolonged administration of synergistic antibiotic combinations. Controversy has arisen regarding optimal therapy (i) when the organism possesses high-level streptomycin resistance, and (ii) when the patient is allergic to penicillin. This study examines these questions in vitro and in a rabbit model of enterococcal endocarditis. The combination of penicillin with either streptomycin or gentamicin increased the rate of bacterial killing in vitro and in vivo when compared with penicillin alone (P < 0.05) when the test strain was relatively susceptible to streptomycin (minimal inhibitory concentration, 128 mug/ml). Only the combination of penicillin and gentamicin was consistently more effective than penicillin alone (P < 0.01) when the test strain was highly resistant to streptomycin (minimal inhibitory concentration > 150,000 mug/ml). The combination of vancomycin and streptomycin was more rapidly bactericidal than vancomycin alone in vitro and in the animal model against the streptomycin-susceptible strain (P < 0.01). The relative rate of in vitro bacterial killing by various antibiotics and combinations was predictive of the efficacy of these drugs in eradicating enterococci from cardiac vegetation in experimental endocarditis.

Protein synthesis in Mycobacterium tuberculosis H37Rv and the effect of streptomycin in streptomycin-susceptible and -resistant strains.

An efficient in vitro amino acid-incorporating system from Mycobacterium tuberculosis H37Rv was standardized. Ribonucleic acid (RNA) isolated from phage-infected M. smegmatis cells served as natural messenger RNA and directed the incorporation of (14)C-amino acids into protein. The effects of various antitubercular drugs and "known inhibitors" of protein synthesis on amino acid incorporation were studied. Antibiotics like chloramphenicol and tetracycline inhibited mycobacterial protein synthesis, though they failed to prevent the growth of the organism. This failure was shown to be due to the impermeability of mycobacteria to these drugs by use of "membrane-active" agents along with the antibiotics in growth inhibition studies. Several independent streptomycin-resistant mutants of M. tuberculosis H37Rv were isolated. Streptomycin inhibited the incorporation of (14)C-amino acids into proteins by whole cells of a streptomycin-susceptible strain by more than 90%, whereas very little or no inhibition was observed in either high-level or low-level streptomycin-resistant strains. In vitro, streptomycin was an effective inhibitor of susceptible strains, whereas in streptomycin-resistant strains the concentration of streptomycin at which half-maximal inhibition was produced varied according to the resistance of whole cells, and there was a correlation between the two. In one low-level streptomycin-resistant mutant, the in vitro amino acid-incorporating system was as sensitive to various concentrations of streptomycin as the parental type, and a possible involvement of a membrane site in the development of low-level resistance was indicated. Streptomycin susceptibility and high-level resistance were shown to be ribosomal in nature.

Binding of dihydrostreptomycin to Escherichia coli ribosomes: characteristics and equilibrium of the reaction.

The binding of dihydrostreptomycin to ribosomes and ribosomal subunits of a number of different Escherichia coli strains was studied, and the Mg(2+) and pH dependence, as well as the effect of salts and polynucleotides, was determined. The only requirement for binding with ribosomes and subunits from susceptible strains was 10 mm Mg(2+). Monovalent salts weakened the binding in a manner similar to the effects on ribonucleic acid secondary structure, and this was antagonized to some extent by increased amounts of Mg(2+). Bound dihydrostreptomycin could be readily exchanged by streptomycin and any antibiotically active derivative, but not by fragments of the antibiotic or any other aminoglycoside. With native (run-off) 70S ribosomes from streptomycin-susceptible strains, the binding was rapid and relatively temperature independent over the range from 0 to 37 C. Polynucleotides did not stimulate the binding. With concentrations of dihydrostreptomycin up to 10(-5)m, greater than 95% of native 70S ribosomes bound exactly 1 molecule of the antibiotic tightly, with a K(diss) for the bound complex at 25 C of 9.4 x 10(-8)m. The following thermodynamic parameters were found for the binding with 70S ribosomes at 25 C:DeltaG degrees = -9.6 kcal/mole, DeltaH degrees = -6.2 kcal/mole, and DeltaS degrees = +11.4 entropy units/mole. Differences in affinity for the antibiotic were found between ribosomes of K-12 strains and those of other E. coli strains. There was insignificant binding to 70S ribosomes or subunits from streptomycin-resistant or -dependent strains, and to 50S subunits from susceptible strains. The binding to 30S subunits from susceptible strains was weaker by an order of magnitude than that to the 70S particle, with a K(diss) at 25 C of 10(-6)m. Polyuridylic acid stimulated this binding slightly but did not influence the affinity of the bound molecule. At antibiotic concentrations above 10(-5)m, streptomycin-susceptible 70S and 30S particles bound additional molecules of the antibiotic, and binding also occurred to ribosomes from streptomycin-resistant and -dependent strains, as well as to 50S subunits from all strains. K(diss) for all of these binding equilibria were [Formula: see text] 10(-4)m. This weaker non-specific binding coincided with the beginning of aggregation phenomena involving the particles, and occurred at sites distinct from the single site which binds the antibiotic tightly. This latter site was completely lost after the one-step mutation to high-level resistance or dependence.

Binding of dihydrostreptomycin to Escherichia coli ribosomes: kinetics of the reaction.

Investigations were carried out on the binding of dihydrostreptomycin to purified (and reassociated) 70S ribosomes and 30S subunits from streptomycin-susceptible strains, and the results were compared with those of similar studies with native (run-off) 70S ribosomes. At 0 C, only a small fraction of purified 70S ribosomes and 30S sub-units bound 1 molecule of the antibiotic tightly, and at a rate comparable to the binding occurring with native 70S ribosomes. At temperatures of 10 C and above, there was a temperature-dependent increase in the extent of antibiotic binding to purified 70S and 30S particles up to a maximum of 1 molecule/ribosomal particle, but the kinetics of binding was slow in comparison to that taking place at 0 C. These and other results suggest that a major fraction of 30S subunits and purified (or reassociated) 70S ribosomes are inactive in binding the antibiotic. This has been localized to an instability of the free 30S subunit, which in solution at 0 C has a half-life of 5 hr or less. Inactive 30S or 70S particles could be thermally activated, with the latter being identical in their streptomycin-binding properties to native 70S ribosomes. The activation kinetics were slow in comparison to the binding kinetics for the antibiotic and were indicative of a conformational change in ribosomal structure. There thus appears to be a reversible transition between active and inactive forms of the ribosomal particles for streptomycin binding, but additional binding sites for the antibiotic are not created by the transitions. The active form of the 30S subunit can be stabilized in the presence of polyuridylic acid, but much more effectively by association with the 50S subunit to form a 70S ribosome. The kinetics of dihydrostreptomycin binding were studied in both directions of the reaction, and the reaction in the direction of binding was found to be several orders of magnitude faster than that of the reverse, or debinding, direction. The kinetics of the exchange of bound dihydrostreptomycin with the free antibiotic were also determined and shown to have rate constants that are very similar to those of the debinding reaction, which is the rate-limiting step. It appears likely that the exchange reaction is proceeding via the same reaction pathway. The temperature dependence of the kinetics of dissociation of the bound complex was much greater than that in the direction of binding and accounted for most of the temperature dependence of the binding equilibrium. From the determined thermodynamic and activation parameters, it appears likely that binding of the antibiotic induces a conformational change in ribosomal structure to one that is less ordered than the native particle. Heterogeneity has been found in the kinetics of binding and of exchange, with a fraction of the 70S population showing slower kinetics for both directions of the reaction.