Drug resistance of Enterococcus faecium clinical isolates and the conjugative transfer of gentamicin and erythromycin resistance traits

Drug resistance and the transferability of resistance were examined in 218 Enterococcus faecium clinical isolates obtained from in-patients of a Japanese university hospital between 1990 and 1999. One hundred and sixty one isolates (73.9%) were drug-resistant and 127 (58.2%) isolates were resistant to two or more drugs. Vancomycin resistant E. faecium (VRE) was not isolated. The transferability of drug-resistance to an E. faecium strain was examined by broth or filter mating. Six (12.5%) of the 48 gentamicin resistance traits, and fifty (50%) of the 101 erythromycin resistance traits were transferred by filter mating. The gentamicin resistance traits of five isolates and the erythromycin resistance traits of four isolates were transferred to the recipient strains by both broth mating and filter mating at a frequency of about 10(-6) and 10(-5) per donor cell, respectively. The five gentamicin resistant strains were shown to harbor pMG1-like plasmids on the basis of their Southern hybridization with pMG1 (65.1 kbp, Gm(r)), which transfers efficiently between enterococci by broth mating. Each of the four erythromycin resistant transconjugants obtained by broth mating harbored a large conjugative plasmid (more than 100 kbp). The plasmids showed no homology with well-characterized enterococcal conjugative plasmids such as pAD1, pPD1, pAM(beta)1, pIP501 and pMG1 by Southern hybridization. Of the erythromycin resistance traits that transferred only by filter mating, it was found that the erythromycin resistance trait was conferred by a 47-kbp transposable element that transferred from the chromosome of the donor strain to different sites within the pheromone responsive plasmid pAD1 (60 kbp) of the recipient strain, suggesting that the erythromycin resistance trait was encoded on a conjugative transposon, which was named Tn950.     

Streptococcus group A resistance to tetracycline: its spread and transduction]

The study on antibiotics resistance of group A streptococci isolated in 1977 showed that the number of the antibiotic resistant strains had significantly increased as compared to the data of 1960. High percentage (53%) of the cultures with multiple resistance was noted. It was observed that the number of the streptococcal cultures resistant to erythromycin and chloramphenicol decreased while the number of the strains resistant to tetracyclines increased. The level of resistance to tetracycline increased more than 2 times from 1960 and in some cases reached 125 and 250 gamma/ml. The wide spread of tetracycline resistance was evident of the presence of the mechanism of the marker transduction. Possible transduction of this feature was studied. Microbe-free phagolysates obtained by induction with UV-light from the strains with multiple antibiotic resistance were used as the donor material in the experiment on transduction. Principal possibility of transducing resistance to tetracycline from 2 donors to 4 recipients at a frequency of 10(-6) was shown.     

Effect of surface-active antiseptics on plasmid transduction of drug resistance and the transducing bacteriophages of staphylococci]

Anionic surface active substances (SAS), such as sodium alkylsulfates, iodonate, sulfanol NP-3 used in subbacteriostatic concentrations lowered at least 100 times the intensity of the erythromycin resistance plasmid in vitro on mixed cultivation of the staphylococcal cells of the donor (strain 8325/11 de) and the recipient (strain 825-1). The cationic SAS, i. e. roc cal, chlorhexidine had no such capacity. The above anionic and cationic SAS had an antiphage effect with respect to the transducing staphylococcal bacteriophages of the serological group B (80, 85, 52A, 53). Such an effect (on the example of sodium alkylsulfates) increased with prolongation of the alkyl radical from C8 to C14. A decrease in the transduction intensity of the erythromycin resistant plasmid in staphylococci was observed in the presence of the anionic SAS either possessing (alkylsulfates, iodonate) or not (sulfonol NP-3) the antiphage activity.     

Transferable lincosamide-macrolide resistance in Bacteroides.

Inter- and intraspecies transfer of resistance to clindamycin, lincomycin, and erythromycin in the strict anaerobe, Bacteroides, is described. This lincosamide-macrolide resistance was found to be specified by a 27 × 10⁶-dalton plasmid, designated pBF4, originally identified in a clinical Bacteroides fragilis isolate. Transfer of this plasmid to a strain of Bacteroides uniformis was demonstrated to occur by a deoxyribonuclease insensitive process which required cell-to-cell contact. Chloroform sterilized donor cell supernatants or filtrates of donor cells did not mediate resistance transfer. Transfer of the antibiotic resistance and pBF4 plasmid deoxyribonucleic acid (DNA) were always coincident. Drug resistant progeny recovered from such matings were able to transfer the pBF4 plasmid and its associated resistance markers to a suitable B.fragilis recipient strain. Compared to interspecies matings, resistance transfer was 100- to 1000-fold greater between isogenic donor and recipient strains, suggesting the possibility of a host controlled restriction-modification system.     

Antibiotic sensitivity of beta-hemolytic Streptococcus group A on a new nutrient medium for the cultivation of streptococci

Sensitivity of 167 strains of beta-hemolytic streptococci of group A was studied with the method of serial dilutions on a solid agar medium for cultivation of streptococci. The medium was developed at the I. I. Mechnikov Research Institute of Vaccines and Sera. It does not require addition of blood or serum. The strains were found to be highly sensitive to penicillin, cephalothin and erythromycin. The number of the strains resistant to tetracycline, streptomycin, gentamycin, levomycetin (chloramphenicol) and ristomycin amounted to 51, 36, 23, 1.8 and 1.8 per cent, respectively. One of the strains (0.6 per cent) was resistant to lincomycin. Strains with multiple resistance were isolated. The necessity of regular control of distribution of antibiotic resistance among staphylococci is indicated.     

Effect of the anthracycline group antibiotics, mitomycin C and bruneomycin, on the transduction of drug resistance in staphylococci]

The effect of various concentrations of antitumor antibiotics, such as carminomycin, rubomycin, adriamycin, mitomycin C and bruneomycin on transduction of erythromycin resistance from the donor strain 8325 P II/de of Staph, aureus to the recipient strain 8325-I in different transduction systems was studied. It was shown that the above antibiotics inhibited the transduction in the systems with constant presence of the drugs. Preliminary treatment of the recipient cells with the drugs in the subbacteriostatic doses did not decrease the transfer frequency. The preliminary treatment of the donor cells resulted in an increase in the phase titer and the transfer frequency in the "preliminary-treated donor + recipient" system.     

Changes in antibiotic sensitivity in strains of Staphylococcus aureus, 1952-78.

Two hundred strains of Staphylococcus aureus isolated from outpatients with infections of the skin and subcutaneous tissues were tested for sensitivity to penicillin, erythromycin, tetracycline, sodium fusidate, methicillin, clindamycin, chloramphenicol, and gentamicin. One hundred and sixty-three (81.5%) of the strains were resistant to penicillin and 16 (8%) resistant to tetracycline. Incidence of resistance to other antibiotics was low. No strain was resistant to chloramphenicol, gentamicin, or methicillin. When compared with results of earlier studies, there was an increase in the incidence of resistance to penicillin and tetracycline, but no appreciable increase in resistance to other antibiotics.     

Transduction of penicillinase production in Staphylococcus epidermidis and nature of the genetic determinant.

Four strains of Staphylococcus epidermidis from clinical sources were capable of serving as donors for the transduction of either penicillinase production, ethidium bromide resistance, or tetracycline resistance. Three typing phages served as transducing phages and, depending upon the combination of transducing phage, donor strain, and recipient strain, the rates of transduction ranged between 10(-5) and 10(-9). In one strain, cotransduction of penicillinase production and ethidium bromide resistance was observed. Although ultraviolet irradiation kinetics indicated that both the tetracycline resistance and the penicillin resistance determinants were located on plasmids, only resistance to tetracycline could be eliminated by growth in the presence of curing agents or at elevated temperature. However, evidence was obtained by agarose gel electrophoretic studies that both the tetracycline resistance and the penicillin resistance determinants are located on separate plasmids in this organism.     

Conjugational plasmid transfer from A, B and H streptococci to N streptococci

Plasmid-mediated resistance to erythromycin and chloramphenicol was successfully transferred from group A, B and H streptococci to group N streptococci by a process akin to conjugation. The results showed that plasmids from streptococcal groups other than N were able to replicate in lactic streptococci as well. The transfer experiments were carried out by using a membrane filter mating technique. Four of the five plasmids used (pSM15346, pSM10419, pIP501, and pEL1) were transferred at frequencies ranging from 10(-1) to 10(-8) transconjugants per donor colony-forming unit. The highest transfer frequencies were obtained when S. pyogenes strain 15346 (pSM15346) served as the donor strain. The identy of transconjugants was verified by testing for the presence of unselected markers of the recipient strains, and both transduction and transformation were ruled out as the mechanisms of transfer.     

Conjugative transfer of glycopeptide and macrolide resistant genes among Enterococci and from Enterococcus faecalis to Staphylococcus aureus

The resistance determinants were transferred from clinical strains of enterococci to Staphylococcus aureus strains. As recipients methicillin-resistant and methicillin-susceptible strains were used and the filter-mating procedure was performed. The transconjugants resistant to erythromycin were obtained in the case of all recipients, in one case the vanA determinant conferring the resistance to vancomycin was transferred together with erythromycin resistance. However the resistance was very unstable and the level was not as high as in the case of Enterococcus faecalis donor strain. The vanA determinant was easily transferred between enterococcal strains by the conjugation and a transfer occurred of vanA alone or together with erythromycin resistance.     

Multiple antibiotic resistance in Rhizobium japonicum.

A total of 48 strains of the soil bacterium Rhizobium japonicum were screened for their response to several widely used antibiotics. Over 60% of the strains were resistant to chloramphenicol, polymyxin B, and erythromycin, and 47% or more of the strains were resistant to neomycin and penicillin G, when tested by disk assay procedures. The most common grouping of resistances in strains was simultaneous resistance to tetracycline, penicillin G, neomycin, chloramphenicol, and streptomycin (25% of all strains tested). The occurrence of multiple drug resistance in a soil bacterium that is not a vertebrate pathogen suggests that chemotherapeutic use of antibiotics is not required for the development of multiple drug resistance.     

Multiple antibiotic resistance in Rhizobium japonicum.

A total of 48 strains of the soil bacterium Rhizobium japonicum were screened for their response to several widely used antibiotics. Over 60% of the strains were resistant to chloramphenicol, polymyxin B, and erythromycin, and 47% or more of the strains were resistant to neomycin and penicillin G, when tested by disk assay procedures. The most common grouping of resistances in strains was simultaneous resistance to tetracycline, penicillin G, neomycin, chloramphenicol, and streptomycin (25% of all strains tested). The occurrence of multiple drug resistance in a soil bacterium that is not a vertebrate pathogen suggests that chemotherapeutic use of antibiotics is not required for the development of multiple drug resistance.     

Antimicrobial resistance of animal and human strains of Enterobacteriaceae in Greece

Two hundred sixty two strains of Enterobacteriaceae from animal and human sources where intermixing and/or spreading is possible were examined for their resistance to 15 antimicrobials frequently used in animal prophylaxis and metaphylaxis. The antimicrobials with the highest proportion of resistant strains from animal sources were amoxicillin 25 mg, colistin sulphate 25 mg, erythromycin 5 mg, penicillin G 10 mg and spectinomycin 10 mg. The same with isolates from human sources where ampicillin 10 mg, amoxicillin 25 mg, colistin sulphate 25 mg, erythromycin 5 mg, neomycin 10 mg, penicillin G 10 mg and spectinomycin 10 mg. Human isolates were resistant to more antimicrobials than animal isolates. Common a high proportion of isolates from both sources were resistant to some antimicrobials. Systematic and better distributed information is needed to address the epidemiological role of veterinary treatment, prophylaxis and metaphylaxis in antimicrobial resistance in Greece.     

Separation of abnormal cell wall composition from penicillin resistance through genetic transformation of Streptococcus pneumoniae.

Compared with most penicillin-susceptible isolates of Streptococcus pneumoniae, penicillin-resistant clinical isolate Hun 663 contains mosaic penicillin-binding protein (PBP) genes encoding PBPs with reduced penicillin affinities, anomalous molecular sizes, and also cell walls of unusual chemical composition. Chromosomal DNA prepared from Hun 663 was used to transform susceptible recipient cells to donor level penicillin resistance, and a resistant transformant was used next as the source of DNA in the construction of a second round of penicillin-resistant transformants. The greatly reduced penicillin affinity of the high-molecular-weight PBPs was retained in all transformants through both genetic crosses. On the other hand, PBP pattern and abnormal cell wall composition, both of which are stable, clone-specific properties of strain Hun 663, were changed: individual transformants showed a variety of new, abnormal PBP patterns. Furthermore, while the composition of cell walls resembled that of the DNA donor in the first-round transformants, it became virtually identical to that of susceptible pneumococci in the second-round transformants. The findings indicate that genetic elements encoding the low affinity of PBPs and the penicillin resistance of the bacteria are separable from determinants that are responsible for the abnormal cell wall composition that often accompanies penicillin resistance in clinical strains of pneumococci.     

Pneumococci resistant to erythromycin.

Susceptibility to erythromycin was determined for all pneumococci isolated in one laboratory from clinical specimens between 1969 and 1977. All 4724 isolates examined prior to October 1973 were susceptible to erythromycin. From October 1973 to December 1977, 64 (0.71%) of 8995 pneumococcus isolates were resistant to erythromycin. The resistant strains were isolated from 38 patients living in six widely separated communities in Alberta. The erythromycin-resistant strains were of nine capsular types, including six that often cause bacteremic disease and five for which resistance to erythromycin has not been reported hitherto. Certain strains of type 33 and of type 15 were highly resistant, the minimum inhibitory concentration (MIC) of erythromycin being 2000 microgram/mL; these strains were also highly resistant to lincomycin and clindamycin. Resistance in strains of other types was much lower, the MIC of erythromycin being 0.6 to 20 microgram/mL, and all but one of these strains were susceptible to lincomycin and clindamycin. All the erythromycin-resistant pneumococci were suspectible to penicillin.     

Geographic diversity and temporal trends of antimicrobial resistance in Streptococcus pneumoniae in the United States

Resistance of Streptococcus pneumoniae to antibiotics is increasing throughout the United States, with substantial variation among geographic regions. We show that patterns of geographic variation are best explained by the intensity of selection for resistance, which is reflected by differences between the proportions of resistance within individual serotypes, rather than by differences between the frequencies of particular serotypes. Using a mathematical transmission model, we analyzed temporal trends in the proportions of singly and dually resistant organisms and found that pneumococcal strains resistant to both penicillin and erythromycin are increasing faster than strains singly resistant to either. Using the model, we predict that by 1 July 2004, in the absence of a vaccine, 41% of pneumococci at the Centers for Disease Control and Prevention (CDC)'s Active Bacterial Core surveillance (ABCs) sites, taken together, will be dually resistant, with 5% resistant to penicillin only and 5% to erythromycin only.     

Transferability of antibiotic resistance determinants in strains of Staphylococcus aureus belonging to different phage groups

A total of 107 donor strains of Staphylococcus aureus isolated from clinical material, with a high incidence of multiresistant strains belonging predominantly to phage group III, were tested for transmission of determinants of resistance to 6 antibiotics using mixed cultures of donor strains and the recipient Staphylococcus aureus strain 5849-fur-r, rif-r. The capability of strains to transfer resistance markers to the recipient was found to depend neither on phage group nor phage type to which the donor strain belonged, but strains possessing multiple resistance to antibiotics effectuated transfers at comparatively higher frequencies.     

Recipient capacity of clinical strains of staphylococci belonging to different phage groups]

The recipient capacity of the strains of Staph. epidermidis and Staph. areus belonging to different phage groups, as well as the possibility of epidemic distribution of the erythromycin resistance marker among the clinical staphyloccal strains on using the defective phage obtained from strain 8325 P IIde was studied. The defective phage P IIde may be the source of epidemic distribution of the drug resistance among the competent strains of Staph. aureus. All erythromycin sensitive strains of Staph. aureus lysed by the phages of groups I and III proved to be competent recipients of the erythromycin resistance marker. The strains of Staph. aureus of phage group II and phage type 80/81, as well as the strains of Staph. epidermidis were not competent recipients under our experimental conditions. It was not possible to transfer the high level of erythromycin resistance (1000 gamma/ml) on transduction to the strains of phage group I with a relatively low level of resistance to this antibiotic (20-50 gamma/ml.     

Effect of an interferon preparation on increasing bacterial sensitivity to antibiotics

Under the effect of human leukocytic alpha-interferon-II for injections there was observed an increase in sensitivity of bacteria to benzylpenicillin, ristomycin, novobiocin, streptomycin, kanamycin, monomycin and erythromycin. A higher antibacterial effect of the penicillin and erythromycin was recorded only with respect to initially resistant strains of staphylococci. For the antibiotic resistant strains of staphylococci the MICs of the penicillin and erythromycin decreased on the average 17.8 and 208 times (p less than 0.001) respectively. The interferon had no effect on sensitivity of bacteria to chloramphenicol.     

Transfer of erythromycin resistance in Staphylococcus aureus.

In the course of an outbreak of enteritis and conjunctivitis, Staphylococcus aureus was isolated from newborn infants. Strains cultured at a later phase of the outbreak differed from those found at the beginning in being resistant to several antibiotics, showing resistance to typing phages and releasing phages of the same lysis spectrum (10(9) p.f.u./ml after heating at 56 degrees C for 2 min). Transduction experiments with a strain and its cell-free lysate showed that inducible erythromycin resistance was transferable to strains isolated at the beginning of the outbreak and to laboratory strains. Plasmid origin of resistance was confirmed by (i) high transduction frequency; (ii) transduction to RN981 rec- mutants; (iii) kinetics of transduction; (iv) elimination of resistance. Mixed culture experiments yielded transductants at high frequency with resistance to erythromycin, streptomycin and tetracycline.