Genetic study of plasmid-associated zonal resistance to lincomycin in Streptococcus pyogenes.

The phenomenon of zonal resistance to lincomycin, which is characteristic of most clinical isolates with lincomycin resistance in Streptococcus pyogenes, has been studied. These strains grow within a defined concentration range of lincomycin (approximately 60 to 200 microgram/ml), or at lincomycin concentrations below the minimal inhibitory concentration for susceptible strains. It is shown that the zonal growth phenomenon is a stable phenotype and results from induction of resistance only within the zonal concentration range of lincomycin. These strains also possess inducible resistance to erythromycin which is nonzonal in character. One-step mutations to constitutive resistance have been isolated which are of two types: constitutive for lincomycin or for erythromycin, but not for both. Those strains with constitutive erythromycin resistance retain their zonal resistance for lincomycin. Mutants doubly constitutive for both lincomycin and erythromycin can be obtained by a second mutational step from either of the singly constitutive mutants. Satellite deoxyribonucleic acid has been shown to be present in the zonal resistant strains. A plasmid, pSM10419, of 14.9 megadaltons, has been isolated from one of the doubly constitutive mutants and used to jointly transform Streptococcus sanguis strain Challis to constitutive resistance to both lincomycin and erythromycin. From this, a multicopy plasmid of reduced size, pSM10 (5.4 megadaltons), which retains its resistance phenotype, has been isolated and mapped with restriction endonucleases HindIII (three sites), EcoRI (one site), KpnI (one site), and HpaI (one site). The staphylococcal plasmid pC221 (2.9 megadaltons; chloramphenicol resistant) has been fused to pSM10 at the EcoRI site resulting in a chimeric plasmid, pSM10221 (8.3 megadaltons), which retains resistance to chloramphenicol, erythromycin, and lincomycin. pSM10 is therefore suggestive as an effective cloning vehicle for the genus Streptococcus.     

In Vitro Development of Resistance to Erythromycin, Other Macrolide Antibiotics, and Lincomycin in Mycoplasma pneumoniae

Mycoplasma pneumoniae was made highly resistant to erythromycin in vitro by serial subculture in broth media containing erythromycin. The resistance developed to erythromycin was 200 mug/ml with the Mac strain, a prototype of M. pneumoniae, and 10 mug/ml with the Fukumura strain, an isolate. The erythromycin resistance was accompanied by cross resistance to other macrolide antibiotics (leucomycin, josamycin, spiramycin, and oleandomycin) and to lincomycin, but there was no resistance to vernamycin B. Resistance to the antibiotics developed in vitro or in vivo was stable after the microorganisms were repeatedly transferred in antibiotic-free media.     

Macrolide resistance in group A streptococci

Two hundred and twenty one Streptococcus pyogenes isolates collected from throat swabs of untreated children with uncomplicated pharyngotonsillitis living in two centres situated in the north of Italy were tested to evaluate their macrolide resistance phenotype. Isolates were also typed for T protein and assayed for opacity factor (OF) and protease production. Resistance to macrolides was found to be similar in the two centres. Fifty-one point two per cent of Torino strains and 43.5% of Pinerolo strains were not inhibited by erythromycin. Resistant strains belonged to one of three phenotypes: CR, constitutive resistance (37.9 and 42.5% in Torino and Pinerolo, respectively); IR, inducible resistance (40.9 and 17. 5%); NR, new resistance phenotype (21.2 and 40%). All the resistant and some of the susceptible strains were analysed by pulsed-field gel electrophoresis and genomic patterns were defined on the basis of band size and number. Five DNA profiles were found among erythromycin-resistant strains: three patterns characterized the NR resistance phenotype and one each the IR and CR phenotypes. The distribution of resistant strains according to their genomic patterns appears to be related to the resistance phenotype and only in some cases to the T serotype of bacteria. We conclude that the S. pyogenes strains analysed are genetically heterogeneous and therefore the high rate of erythromycin resistance observed is not caused by the spread of a single clone nor is it related to a particular serotype.     

Macrolide-lincosamide-streptogramin resistance patterns in Clostridium perfringens from animals

Different patterns of resistance against commonly used macrolide, lincosamide, and streptogramin antibiotics were found in Clostridium perfringens of animal origin. The patterns were designated as (i) macrolide-lincosamide-streptogramin group B generalized resistance, (ii) macrolide-lincosamide generalized resistance, (iii) macrolide-lincosamide inducible resistance, and (iv) macrolide-lincosamide-streptogramin low-level generalized resistance. The strains of the fourth pattern were able to inactivate pristinamycin and virginiamycin. The macrolide-susceptible strains showed a bimodal distribution of lincomycin and clindamycin susceptibility levels. The susceptible strains were inhibited by 0.25 micrograms of lincomycin per ml and 0.03 micrograms of clindamycin per ml. The low-level resistant strains were inhibited at concentrations of 2 to 4 micrograms of lincomycin per ml and 0.5 to 2 micrograms of clindamycin per ml.     

Drug Resistance in Streptococcus pyogenes Isolated in Japan (1974–1975)

Drug resistance of Streptococcus pyogenes strains isolated during 1974 and 1975 in various districts in Japan were surveyed and compared with an earlier survey of 1970 to 1973. Of 1,021 strains, tetracycline-, macrolide antibiotic-, lincomycin-, and chloramphenicol-resistant strains were demonstrated at frequencies of 80.3, 62.3, 60.8, and 57.9%, respectively. Distinct group resistances to penicillin and aminoglycoside antibiotics could not be identified among the strains examined. It was characteristic that quadruple and triple resistances were manifested among the strains resistant to macrolide antibiotics, lincomycin, tetracycline, and chloramphenicol, and they were confined to the T-type 12. The emergence of multiply resistant streptococcal strains was due mostly to the rapid increase in isolation frequency of macrolide antibiotic- or macrolide antibiotics-lincomycin-resistant strains.     

Erythromycin Resistance Genes in Group A Streptococci in Finland

Streptococcus pyogenes isolates (group A streptococcus) of different erythromycin resistance phenotypes were collected from all over Finland in 1994 and 1995 and studied; they were evaluated for their susceptibilities to 14 antimicrobial agents (396 isolates) and the presence of different erythromycin resistance genes (45 isolates). The erythromycin-resistant isolates with the macrolide-resistant but lincosamide- and streptogramin B-susceptible phenotype (M phenotype) were further studied for their plasmid contents and the transferability of resistance genes. Resistance to antimicrobial agents other than macrolides, clindamycin, tetracycline, and chloramphenicol was not found. When compared to our previous study performed in 1990, the rate of resistance to tetracycline increased from 10 to 93% among isolates with the inducible resistance (IR) phenotype of macrolide, lincosamide, and streptogramin B (MLS_B) resistance. Tetracycline resistance was also found among 75% of the MLS_B-resistant isolates with the constitutive resistance (CR) phenotype. Resistance to chloramphenicol was found for the first time in S. pyogenes in Finland; 3% of the isolates with the IR phenotype were resistant. All the chloramphenicol-resistant isolates were also resistant to tetracycline. Detection of erythromycin resistance genes by PCR indicated that, with the exception of one isolate with the CR phenotype, all M-phenotype isolates had the macrolide efflux (mefA) gene and all the MLS_B-resistant isolates had the erythromycin resistance methylase (ermTR) gene; the isolate with the CR phenotype contained the ermB gene. No plasmid DNA could be isolated from the M-phenotype isolates, but the mefA gene was transferred by conjugation.     

Isolation and Characterization of Multiply Antibiotic-Resistant Clostridium perfringens Strains from Porcine Feces

Multiply antibiotic-resistant strains of Clostridium perfringens were isolated from porcine feces. Strains that were resistant to tetracycline, erythromycin, clindamycin, and lincomycin were isolated, but no penicillin- or chloramphenicol-resistant strains were obtained. Typical minimal inhibitory concentrations for resistant strains were 16 to 64 mug of tetracycline per ml, 64 to >128 mug of erythromycin per ml, >/=128 mug of lincomycin per ml, and 16 to 128 mug of clindamycin per ml. Resistance to erythromycin was always associated with resistance to lincomycin and clindamycin. Minimal inhibitory concentrations were determined for 258 strains from six farms that used antibiotics in their feeds and 240 strains from five farms that did not use antibiotics. The results show that 77.9 and 22.7% of the strains from the former farms were resistant to tetracycline and erythromycin-clindamycin-lincomycin, respectively. The comparable data from the latter farms were 25.0 and 0.8%, respectively. Agarose gel electrophoresis failed to reveal a plasmid band that was common to the resistant strains but absent in the susceptible strains. Attempts to transfer tetracycline, erythromycin, and clindamycin resistance from one strain, CW459, were not successful. Antibiotic-susceptible mutants were not isolated from this strain, despite the use of a variety of curing agents.     

Rosamicin: Evaluation In Vitro and Comparison with Erythromycin and Lincomycin

Rosamicin is a new macrolide antibiotic produced by Micromonospora rosaria. It shares certain chemical and biological characteristics with erythromycin. Activity against gram-positive strains was assayed by broth dilution and compared to that of erythromycin and lincomycin. Rosamicin was bacteriostatic and inhibited most strains of Staphylococcus aureus, Staphylococcus epidermidis, enterococci, viridans streptococci, and group A streptococci in concentrations of 0.02 to 4.0 mug/ml. Results were similar for erythromycin and for lincomycin (excluding enterococci). Cross-resistance of gram-positive organisms to these three antimicrobial agents was incomplete. Rosamicin was more active than erythromycin against Enterobacteriaceae and Pseudomonas at pH 7.2. Alkalinization of the medium enhanced the activity of both rosamicin and erythromycin; however, rosamicin was still more active than erythromycin against all gram-negative strains at pH 7.6 and 8.0. In view of the high degree of in vitro activity of rosamicin against gram-positive organisms, lack of complete cross-resistance with erythromycin and lincomycin, and the greater activity of rosamicin than erythromycin against gram-negative organisms, further investigation of this macrolide is warranted.     

Susceptibility of Clostridium ramosum to antimicrobial agents

The susceptibility of 49 strains of Clostridium ramosum to 10 antibiotics was determined by agar dilution and disk diffusion tests. Results showed that, among the anaerobes, C. ramosum is second only to Bacteroides fragilis in its resistance to antimicrobial agents. All strains were susceptible to penicillin, carbenicillin, chloramphenicol, vancomycin, and metronidazole at readily achievable blood levels. Most strains (83%) were susceptible to erythromycin. There was a high level of resistance to clindamycin in 16% of the strains. All isolates were resistant to rifampin and gentamicin, and most were resistant to lincomycin. Assessment of susceptibility by measurement of inhibition zone diameters with disk diffusion tests was not satisfactory.     

Resistance of Group A Beta-Hemolytic Streptococci to Lincomycin and Erythromycin

Ten (0.05%) of 18,628 strains of Streptococcus pyogenes isolated from clinical specimens in the 3 years 1968 to 1970 were resistant to lincomycin and erythromycin. All 10 strains were highly resistant to lincomycin, having minimal inhibitory concentration (MIC) values of 200 mug/ml. There were two degrees of resistance to erythromycin: four strains were highly resistant, having MIC values of 200 mug or more/ml; and six strains showed slight resistance, MIC values being 0.78 to 1.56 mug/ml. There was no known epidemiological relationship between any of the patients infected with the resistant strains, which belonged to a variety of T serotypes. A zonal pattern of resistance to lincomycin occurred in four strains, all of which were only slightly resistant to erythromycin. After incubation for 24 hr in a twofold dilution series of lincomycin in broth, the strains grew in 0.05 mug or less/ml and in 50 and 100 mug/ml, but not in intermediate concentrations. Tests in agar indicated that the bacterial population of one strain, but not of the other three, was homogeneous in respect to its ability to grow readily in low and high, but not in intermediate, concentrations. The zone phenomenon is of significance in the clinical laboratory, since unawareness of it might result in a highly resistant strain being regarded as susceptible to lincomycin in tube or plate MIC tests that do not include sufficiently high concentrations of lincomycin.     

Bacteriostatic and Bactericidal Activities of Various Antibiotics Against Bacteroides fragilis

The minimal inhibitory concentrations and minimal bactericidal concentrations of seven antibiotics were determined by broth dilution tests for 50 clinical isolates of Bacteroides fragilis. Chloramphenicol, erythromycin, lincomycin, clindamycin, and rifampin inhibited 80 to 100% of the strains at concentrations attainable in the serum. Penicillin and tetracycline were less effective, inhibiting 4 and 40% of the strains, respectively, at concentrations attainable in the serum. The strains exhibited a bimodal distribution with tetracycline but a very narrow pattern of susceptibility with chloramphenicol. Bactericidal concentrations were 4- to 128- fold higher than minimal inhibitory concentrations for all antibiotics; only clindamycin showed bactericidal activity at levels attainable in serum. Clindamycin was significantly more effective than lincomycin as determined by tests of inhibitory and bactericidal activity. The distinct patterns of susceptibility of B. fragilis may be used for the preliminary selection of antimicrobial therapy.     

Macrolide-lincosamide-streptogramin B (MLS) resistance in cutaneous propionibacteria: definition of phenotypes

Erythromycin-resistant propionibacteria isolated from the skin of antibiotic-treated acne patients were found to express four different patterns of resistance to a set of eight MLS antibiotics. The majority of isolates (48/89 strains) were constitutively resistant to 14- and 16-membered ring macrolide, lincosamide and streptogramin B-type antibiotics. MICs of josamycin (0.5-16 mg/l) and spiramycin (0.5-128 mg/l) were lower than those of erythromycin, oleandomycin and tylosin (64 to less than 512 mg/l). Two strains of Propionibacterium granulosum exhibited inducible MLS resistance. Both 14- and 16-membered ring macrolides and virginiamycin S induced clindamycin resistance in these strains. Fifteen isolates demonstrated a similar phenotype to the inducible strains but were non-inducible by erythromycin. A fourth group of strains demonstrated high level resistance to all five macrolides tested (MIC greater than or equal to 128 mg/l) but were sensitive to virginiamycin S. The phenotype displayed by these strains is not compatible with the expression of methylase genes as currently known, nor with the action of an erythromycin esterase which hydrolyses only 14-membered ring macrolides. The resistance patterns of 12 isolates could not be classified into any of the above phenotypic classes. Therefore, the majority of erythromycin resistant propionibacteria express MLS resistance which is phenotypically similar to that coded for by several well characterized RNA methylase (erm) genes.     

Epidemiology of Antibiotic and Heavy Metal Resistance in Bacteria: Resistance Patterns in Staphylococci Isolated from Populations in Iraq Exposed and Not Exposed to Heavy Metals or Antibiotics

Staphylococci were isolated from rural and urban populations in Iraq, which were not known to be exposed to either heavy metals or antibiotics. The antibiotic and heavy metal resistance patterns of these strains were analyzed in both mannitol-fermenting and nonfermenting strains. Over 90% of the strains were resistant to at least one of the following antibiotics: penicillin, chloramphenicol, erythromycin, tetracycline, cephalothin, lincomycin, or methicillin. In general, mannitol-fermenting strains were resistant to penicillin and cupric ions. Mannitol-negative strains were more frequently associated with mercuric ion and tetracycline resistance. Although resistance to penicillin and tetracycline can coexist, the combination of penicillin resistance and tetracycline resistance usually occurred in mannitol-negative strains. The possibility of selection of heavy metal-resistant strains due to exposure to toxic levels of methylmercury was examined. No significant increase in mercuric ion-resistant strains of staphylococci or Escherichia coli were detected in exposed populations as compared to control groups. The possible reasons for this result are discussed.     

Mechanism of Penicillin-Erythromycin Synergy on Antibiotic-Resistant Staphylococcus aureus

Clinically isolated strains of Staphylococcus aureus that are inducibly resistant to both erythromycin and penicillin were susceptible to a combination of the two antibiotics. The synergistic effect of the combination results from an inhibition of penicillinase induction by erythromycin, sparing penicillin and allowing this drug to inhibit growth. When resistance to erythromycin is constitutive rather than inducible, the combination is no longer synergistic.     

Usefulness of an Erythromycin-Resistant Strain of Mycoplasma pneumoniae for the Fermentation-Inhibition Test

The fermentation-inhibition (FI) test for Mycoplasma pneumoniae was performed with two strains of M. pneumoniae, one susceptible to erythromycin and one highly resistant to erythromycin with cross-resistance to other macrolide antibiotics. Serum titers in children with M. pneumoniae pneumonia who received no antibiotic were similar with the two strains. Children with atypical pneumonia with a transient rise in the FI titer with the susceptible strain proved to have received erythromycin at the time of the rise. They showed no rise in the FI titer done with the erythromycin-resistant strain. Oral administration of erythromycin regularly elevated the serum FI titer when the test was done with the susceptible strain. Use of the resistant strain in the test eliminated this false elevation. Tetracycline and chloramphenicol did not elevate the titer, even with the susceptible strain. Use of the strain of M. pneumoniae resistant to erythromycin provides a true FI antibody serum titer, avoiding the influence of antibiotics.     

In vitro exposure to macrolide antibiotics in <Emphasis Type="Italic">Helicobacter pylori</Emphasis> strains isolated from children

Clarithromycin resistance of Helicobacter pylori is a serious problem in eradication therapy. We investigated whether the use of maclorides (clarithromycin, erythromycin, and azithromycin) induces clarithromycin resistance in the organism. Twenty H. pylori strains were isolated from pediatric patients with gastrointestinal symptoms. The minimum inhibitory concentrations (MICs) of each macrolide antibiotic were determined by the Etest. Among these, 17 strains susceptible to macrolide antibiotics were used for the in vitro induction of drug resistance. In each of these 17 strains of H. pylori, 30-day exposure to clarithromycin in experiments for in vitro induction did not change the MIC of any antibiotic, nor did it induce either the A2143G or the A2144G mutation in the 23S rRNA gene. These results suggest that the use of macrolide antibiotics does not induce clarithromycin resistance in H. pylori by 23S rRNA gene mutation.     

Induction of erm(C) expression by noninducing antibiotics

Ketolides, which represent the newest macrolide antibiotics, are generally perceived to be noninducers of inducible erm genes. In the study described in this paper we investigated the effects of several macrolide and ketolide compounds on the expression of the inducible erm(C) gene by Escherichia coli cells. Exposure to 14-member-ring macrolide drugs and to azithromycin led to a rapid and pronounced increase in the extent of dimethylation of Erm(C) target residue A2058 in 23S rRNA. When cells were incubated with subinhibitory concentrations of ketolides, the extent of A2058 dimethylation was also increased, albeit to a lower level and with kinetics slower than those observed with macrolides. The induction of erm(C) expression by ketolides was further confirmed by using a reporter construct which allows the colorimetric detection of induction in a disc diffusion assay. Most of the ketolides tested, including the clinically relevant compounds telithromycin and cethromycin, were able to induce the reporter expression, even though the induction occurred within a more narrow range of concentrations compared to the concentration range at which induction was achieved with the inducing macrolide antibiotics. No induction of the reporter expression was observed with 16-member-ring macrolide antibiotics or with a control drug, chloramphenicol. The deletion of three codons of the erm(C) leader peptide eliminated macrolide-dependent induction but left ketolide-dependent induction unchanged. We conclude that ketolides are generally capable of inducing erm genes. The narrow range of ketolide inducing concentrations, coupled with the slow rate of induction and the lower steady-state level of ribosome methylation, may mask this effect in MIC assays.     

Antibiotic sensitivity of ribosomes from wild-type and clindamycin resistant Bacteroides vulgatus strains.

The sensitivity to different antibiotics of in-vitro polyuridylic acid-dependent polypeptide synthesizing system from Bacteroides vulgatus RYC18F6 and two clindamycin-resistant derivatives was studied. The ribosomes from the resistant strains were not affected by concentrations of up to 0.1 mM clindamycin and lincomycin. In contrast, streptogramin B was found to cause strong stimulation of the clindamycin-resistant polymerizing systems. The modified ribosomes from the resistant strains were more sensitive to other antibiotics like sparsomycin and chloramphenicol. The data indicate that resistance in these B. vulgatus mutant strains is due to alteration of the ribosome structure.     

Susceptibility of Pneumococci and Haemophilus influenzae to Antibacterial Agents

Strains of Diplococcus pneumoniae and Haemophilus influenzae were tested for susceptibility to numerous antibiotics by a twofold agar dilution method using an inocula replicator. Undiluted, fully grown broth cultures were used as inocula for both species, and cultures of pneumococci diluted 1:1,000 were also tested. The antibiotics included most of those in common use in the United States as well as some chemical modifications recently approved and others that are under investigation. The most striking aspect of the results was the marked susceptibility of the pneumococci to all the antibiotics tested except the polymyxins and most of the aminoglycoside antibiotics, although some new aminoglycosides were active in quite low concentrations. Some of the strains of pneumococci were of decreased susceptibility to penicillin G (minimal inhibitory concentrations, 0.2 to 0.4 mug/ml), but none were tetracycline resistant, although such strains had been reported previously from this laboratory. The strains of H. influenzae, which were all serologically nontypable, exhibited different patterns of susceptibility to the groups of antibiotics and to the individual chemically related ones. None of these strains (isolated early in 1972) were ampicillin resistant. The most active agents against H. influenzae were: carbenicillin and ampicillin, analogues related to each of them, rifampin, chloramphenicol, and the polymyxins. However, the tetracycline analogues other than tetracycline, some aminoglycosides, notably tobramycin, kanamycin, gentamicin, and verdamicin, erythromycin, and some new lincomycin analogues were also active in low concentrations. Trimethoprim alone was highly active, and in combination with sulfamethoxazole it was even more active and synergistic against strains of both D. pneumoniae and H. influenzae.     

In-vitro activities of 14-, 15- and 16-membered macrolides against gram-positive cocci.

The in-vitro activities of the 14-membered macrolides erythromycin, dirithromycin, roxithromycin, clarithromycin, the 15-membered compound azithromycin and the 16-membered macrolides (16 MM) josamycin, spiramycin and midecamycin acetate (MOM) have been compared against staphylococci, enterococci and streptococci. Results have been analysed separately according to the sensitivity status of the tested strains to erythromycin, namely sensitive (S), inducibly resistant (IR) or constitutively resistant (CR). 14- and 15-membered macrolides were active only against S strains; the order of potency in vitro was clarithromycin = erythromycin greater than azithromycin = roxithromycin greater than dirithromycin. The 16 MM were slightly less active against S strains than were the 14- and 15-membered compounds, and inhibited most IR strains; MOM and josamycin were about twice as potent as spiramycin. IR and S Staphylococcus aureus strains were equally sensitive to 16 MM, while IR strains of coagulase-negative staphylococci were less sensitive than were S strains. All CR strains of S. aureus were resistant to 16 MM, as were most of the other CR strains. However, 5/21 CR coagulase-negative staphylococci and 2/20 CR enterococci tested were sensitive to 16 MM. The seven CR strains showing anomalous sensitivity to the 16 MM (five Staphylococcus haemolyticus and two enterococci) were only 'moderately resistant' to erythromycin (MIC 8-64 mg/L), while all the other CR strains were 'highly resistant' (MIC greater than 128 mg/L). These results indicate that it may be difficult to predict the sensitivity of Gram-positive cocci to 16 MM, and therefore individual sensitivity testing to specific compounds is essential.