Mechanisms of R Factor R931 and Chromosomal Tetracycline Resistance in Pseudomonas aeruginosa

The mechanism of tetracycline resistance mediated by R931 (a Pseudomonas aeruginosa R factor not yet successfully transferred to Escherichia coli recipients) was examined. In strain 931 (R931) (minimal inhibitory concentration [MIC] 200 mug/ml) significant tetracycline uptake did not occur until 100 mug of tetracycline per ml was included in uptake studies. The introduction of R931 into strain 280 resulted in a significant decline in (3)H-tetracycline uptake. In both strains 931 (R931) and 280 (R931), a further reduction in tetracycline uptake resulted from pre-incubation with 1 mug of tetracycline per ml. Tetracycline resistance in R(-)P. aeruginosa strains 1731, 1885, and 494, considered to be of chromosomal origin, was associated with a lack of tetracycline uptake until the MIC of the strain was obtained. No evidence of tetracycline inactivation or ribosomal resistance was detected in R(-) or R(+) strains. The MIC for R(-) strains was generally about 25 mug/ml and that for R(+) strains was 75 to 200 mug/ml.     

Resistance of Pseudomonas aeruginosa to gentamicin and related aminoglycoside antibiotics

This study was undertaken to investigate biochemical, genetic, and epidemiological aspects of resistance to aminoglycoside antibiotics among 650 consecutive isolates of Pseudomonas aeruginosa from Parkland Memorial Hospital, Dallas, Tex. In 364 strains, minimal inhibitory concentrations were 25 mug/ml or greater for gentamicin (G), tobramycin (T) or kanamycin (K). Four patterns of resistance were noted: (A) G, T, K (four strains), (B) G, K (23 strains), (C) T, K (one strain), and (D) K (336 strains). Gentamicin acetyltransferase (GAT) activities were associated with resistance to gentamicin in strains of groups A and B, whereas kanamycin phosphotransferase activity was found in strains of group D. The GAT from group B strains acetylates both gentamicin and tobramycin. Resistance to gentamicin and susceptibility to tobramycin may reflect the fact that the K(m)'s for tobramycin (25 to 44 mug/ml) of GAT activities in these group B strains are much greater than the K(m)'s for gentamicin (1.9 to 2.7 mug/ml) and exceed the minimal inhibitory concentrations for tobramycin (1.25 to 7.5 mug/ml). GAT from strains of group A was associated with resistance to G, T, and K. Gentamicin acetyltransferases can be distinguished by their specificities for aminoglycoside substrates. The substrate specificity of GAT from group B strains is similar to that reported for GAT(I), but the specificity of GAT from group A strains differs from those described for GAT(I) and GAT(II). Conjugal transfer of gentamicin or tobramycin resistance from our strains of P. aeruginosa to various potential recipient strains was not observed. Pyocin typing showed that many, but not all, of the strains resistant to gentamicin were similar, and retrospective epidemiological investigation revealed that these strains were isolated almost exclusively from patients in the adult and pediatric burn intensive care units and geographically continguous areas of the hospital.     

Activity of Lividomycin Against Pseudomonas aeruginosa: Its Inactivation by Phosphorylation Induced by Resistant Strains

The antibacterial activity and enzymatic inactivation of lividomycin, a new aminoglycosidic antibiotic, were studied with 13 strains of Pseudomonas aeruginosa. The minimal inhibitory concentration of lividomycin was 12.5 to 25 mug/ml, and three strains were resistant to high concentrations of lividomycin (more than 200 mug/ml). It was found that P. aeruginosa TI-13 and K-11, highly lividomycin-resistant strains of clinical origin, strongly inactivated the drug. The third resistant strain, Km-41/R, was developed in vitro. Unlike the other resistant strains, Km-41/R, was developed in vitro. Unlike the other resistant strains, Km-41/R did not inactivate the drug, indicating that different mechanisms were involved in lividomycin resistance. By use of a cell-free extract from P. aeruginosa TI-13, the inactivation of lividomycin was found to be caused by the formation of a monophosphorylated product of the drug.     

Characteristics of R931 and Other Pseudomonas aeruginosa R Factors

R factors were detected in 3.3% of 233 hospital isolates of Pseudomonas aeruginosa using P. aeruginosa recipients in conjugations. Transferred markers included streptomycin, tetracycline, and sulfonamide resistance. Gentamicin resistance was transferred from two strains previously shown to acetylate gentamicin. A group of R factors exemplified by R931 were characterized by failure to transfer to Escherichia coli recipients. Such R factors formed a single compatibility group when examined in a P. aeruginosa recipient. Other P. aeruginosa R factors, including RP4, showed stable coexistence with the R931 group. It is proposed that RP4 and similar R factors be members of the P-1 compatibility group and that R931, R3108, R209, and R130 be members of a group termed P-2. The buoyant densities of all R factors examined were similar, about 1.716 to 1.719 g/cm(3). The content of R-factor deoxyribonucleic acid (DNA) relative to the total DNA varied among the different R factors, ranging from about 18 +/- 2% in log-phase cells of 931 (R931) to undetectable for 679 (R679). However, R679, which transferred from strain 679 at extremely low and irregular frequencies to an E. coli host, was shown to represent about 4% R-factor DNA in that host. The relative DNA content of R931 appeared to decline in the stationary growth phase of 931 (R931) or 280 (R931). R931 covalently closed circular DNA was isolated by ethidium bromide-CsCl gradient centrifugation and examined by electron microscopy. Two major molecular distributions existed, having contour lengths of 0.5 and 12.4 mum. The molecular weights were estimated to be 10(6) and 25 x 10(6). Both molecules were under relaxed replication control. R factor R931 exists as a naturally occurring high-frequency transfer system in P. aeruginosa strains 931 and 1310. However, in strain 280 it acts as if subject to fertility repression. Other members of the P-2 compatibility group also are high-frequency transfer systems in the natural host and in strain 1310. RP4 is restricted from recipient strain 1310. Some additional recipient effects were noted in that strains 1310 or 280 sometimes differed in recipient effectiveness with a given donor. Agglutination reactions with absorbed antiserum were able to distinguish between two members of the same R-factor compatibility group, R931 and R3108.     

Interaction of clindamycin and gentamicin in vitro

The minimal inhibitory concentrations of clindamycin and gentamicin alone and in combinations were determined by a microdilution method for 163 aerobic, facultative, and anaerobic clinical isolates. All 77 strains of Staphylococcus aureus, Diplococcus pneumoniae, Streptococcus pyogenes, and anaerobic bacteria (except for three strains of Clostridium) were inhibited by 1.6 mug or less of clindamycin per ml. Gentamicin did not interfere with the activity of clindamycin within the range of concentrations tested (0.1 to 100 mug/ml); for some strains combinations were synergistic. Sixty-two (94%) of 66 strains of Enterobacteriaceae and Pseudomonas aeruginosa were inhibited by 6.2 mug or less of gentamicin per ml. Combinations of clindamycin and gentamicin were indifferent for 29 strains and synergistic for 33 strains. All 20 strains of enterococcus, three strains of Clostridium, three strains of Escherichia coli, and one strain of Proteus rettgeri were resistant to both clindamycin (minimal inhibitory concentration greater than 3.1 mug/ml) and gentamicin (minimal inhibitory concentration greater than 6.2 mug/ml). Combinations of clindamycin and gentamicin were indifferent for 16 and synergistic for 11 of the resistant strains. Except for clindamycin-sensitive isolates, synergy was usually observed only at concentrations of one or both drugs which are not readily obtainable in vivo. Antagonism was never observed.     

In Vitro Comparison of Kanamycin, Kanendomycin, Gentamicin, Amikacin, Sisomicin, and Dibekacin Against 200 Strains of Pseudomonas aeruginosa

The antimicrobial activity of kanamycin, kanendomycin, gentamicin, amikacin, sisomicin, and dibekacin against 200 strains of Pseudomonas aeruginosa was compared. Dibekacin was found to be the most active against the tested organisms, whereas the other aminoglycoside antibiotics fell in the following order of diminishing antibacterial potency: amikacin, sisomicin, gentamicin, kanendomycin, and kanamycin. Seven strains showed high-level resistance to gentamicin (minimal inhibitory concentration, 400 mug/ml), and two of them were also resistant to amikacin and sisomicin (minimal inhibitory concentration, 75 mug/ml). The minimal inhibitory concentration of dibekacin for these seven strains was 0.625 mug/ml.     

Antibacterial Activity of Apalcillin (PC-904) Against Gram-Negative Bacilli, Especially Ampicillin-, Carbenicillin-, and Gentamicin-Resistant Clinical Isolates

Apalcillin (PC-904) is active against carbenicillin- and ampicillin-resistant strains of gram-negative bacilli. Among Pseudomonas aeruginosa strains highly resistant to carbenicillin (>/=3,200 mug/ml), half of them were susceptible to PC-904 at a concentration of 50 to 1,600 mug/ml. The minimal inhibitory concentration of PC-904 against P. aeruginosa strains resistant to carbenicillin (400 to 1,600 mug/ml) ranged from 3.1 to 25 mug/ml. Ampicillin- and carbenicillin-resistant Enterobacteriaceae strains were similarly susceptible to PC-904. However, drug resistance to PC-904 was already apparent among some strains of P. aeruginosa, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, P. vulgaris, and P. morganii, recently isolated in Japan; i.e., 4, 35, 32, 4, 6, and 14% of strains isolated were resistant. PC-904 was more active, on the other hand, than ampicillin and carbenicillin against antibiotic-susceptible Enterobacteriaceae and also showed high activity against most species of Pseudomonadaceae, especially P. cepacia and P. aeruginosa. The minimum inhibitory concentrations of PC-904 were greatly affected by inoculum size when the organisms tested were strains producing large amounts of beta-lactamase.     

Comparative Activity of Tobramycin, Amikacin, and Gentamicin Alone and with Carbenicillin Against Pseudomonas aeruginosa

The effect of gentamicin against 130 clinical isolates of Pseudomonas aeruginosa was compared with that of two investigational aminoglycoside antibiotics, tobramycin and amikacin. Minimal inhibitory concentration data indicated that, on a weight basis, tobramycin was two to four times as active as gentamicin against most isolates. However, 14 of 18 organisms highly resistant to gentamicin (>/=80 mug/ml) were also highly resistant to tobramycin. Amikacin was the least active aminoglycoside on a weight basis, but none of the isolates were highly resistant to this antibiotic. When therapeutically achievable concentrations were used, adding carbenicillin to gentamicin or to tobramycin enhanced inhibitory activity against those isolates susceptible (相似文献   

Synergy studies with Pseudomonas aeruginosa resistant to gentamicin and/or carbenicillin.

The predictability of synergy with strains of Pseudomonas aeruginosa highly resistant to gentamicin in combination with carbenicillin has been controversial. 30 clinical isolates of P. aeruginosa resistant to gentamicin and/or carbenicillin were tested by checker-board technique. 14 were found to be highly resistant to gentamicin (minimal inhibitory concentration MIC greater than or equal to 128 microgram/ml) and/or carbenicillin (MIC greater than or equal to 512 microgram/ml). Of these 14, 4 isolates showed synergy. 10 of 16 isolates with moderate resistance demonstrated synergy. It is concluded that the level of resistance to gentamicin with P. aeruginosa cannot be used in predicting whether synergy will occur.     

Lipopolysaccharide changes in impermeability-type aminoglycoside resistance in Pseudomonas aeruginosa.

Clinical isolates of Pseudomonas aeruginosa were examined for the basis of impermeability-type aminoglycoside resistance. Two apparently related burn isolate strains with high-level (strain 8803) and low-level (strain 13934) gentamicin resistance each had a plasmid. Transformation of the plasmid from either strain to P. aeruginosa PAO503 resulted in low-level gentamicin resistance. No mechanism for this resistance could be determined. Low-level gentamicin and streptomycin resistance from strain 8803 (but not 13934) was transduced with phage E79.tv2 to PAO503 without transfer of plasmid DNA. Transductants like strain 8803 showed absence or reduction of the lipopolysaccharide (LPS) "ladder" pattern of PAO503, had a change in chemical composition of LPS, and, like strain 8803, had a reduced capability to accumulate streptomycin. Comparison of the resistant clinical isolates 8803 and P10 with the apparently related but less-resistant strains 13934 and P10R, respectively, showed the latter strains had LPS ladder patterns and the former strains did not. Strain 8803 had normal outer membrane protein profiles, electron transport components, and transmembrane electrical potential relative to PAO503 and has been previously shown to have no detectable gentamicin-modifying enzymes and normal protein synthesis. We conclude that low-level impermeability-type aminoglycoside resistance in P. aeruginosa results from conversion of smooth LPS to superficial or deeper rough LPS phenotypes. High-level resistance apparently results from a plasmid-specified, but as yet unknown, mechanism combined with the preceding change in LPS structure.     

Comparison of the in vitro activity of BL-P1654 with gentamicin and carbenicillin against Pseudomonas aeruginosa

The in vitro activity of 6-[d-alpha-(3 guanylureido)-phenylacetamido]-penicillanic acid (BL-P1654) was evaluated against 117 clinical isolates of Pseudomonas aeruginosa, many of which were known to be resistant to both gentamicin and carbenicillin. BL-P1654 was two to eight times more active than carbenicillin against P. aeruginosa. However, all 28 highly carbenicillin-resistant isolates (minimal inhibitory concentration [MIC] > 500 mug/ml) were also highly resistant to BL-P1654. When an MIC of 32 mug/ml or less and a zone of inhibition of 12 mm or more by a 10-mug disk were used as criteria indicating susceptibility to BL-P1654, the false-resistance rate by the disk test was 10.6% and the false-susceptibility rate was 4.2%. The combination of BL-P1654 and gentamicin was synergistic against 45 of 70 isolates of P. aeruginosa tested, but synergism was demonstrated against only 4 of 24 highly gentamicin-resistant (MIC > 63 mug/ml), 1 of 12 highly BL-P1654-resistant (MIC > 250 mug/ml) isolates, and none of nine isolates highly resistant to both of these antibiotics.     

Studies on the Susceptibility of 150 Consecutive Clinical Isolates of Pseudomonas aeruginosa to Tobramycin, Gentamicin, Colistin, Carbenicillin, and Five Other Antimicrobials

The in vitro susceptibilities of 150 clinical isolates of Pseudomonas aeruginosa to the aminoglycoside tobramycin and eight other antimicrobials were evaluated. The Food and Drug Administration standardized disk diffusion method showed that tobramycin inhibited 100% of the 150 isolates with gentamicin inhibiting only 90.7%. The difference between colistin (97.3%) and tobramycin was less marked. Carbenicillin (87.3%) was found to be slightly less active than gentamicin. Only a small percentage of the isolates were inhibited by the other drugs used. By using a minimal inhibitory concentration of 4 mug/ml in brain heart infusion broth as the level of susceptibility, it was found that only three of the isolates showed resistance to tobramycin, whereas 20 of the isolates were resistant to gentamicin. Approximately half of the isolates were inhibited by 0.5 mug/ml or less of tobramycin, whereas 1.5 mug of gentamicin per ml was required to inhibit 50% of susceptible strains.     

Molecular characterization of highly gentamicin-resistant Enterococcus faecalis isolates lacking high-level streptomycin resistance.

Antimicrobial susceptibilities and DNA contents were analyzed for six clinical isolates of Enterococcus faecalis that had high-level resistance to gentamicin (MIC > 2,000 micrograms/ml) but not streptomycin and were obtained from patients in diverse geographic areas. Contour-clamped homogeneous electric field electrophoresis of genomic DNA showed all isolates to be different strains. Gentamicin resistance was transferred from four isolates to plasmid-free enterococcal recipients in filter matings. Restriction enzyme analysis of transconjugants showed distinct gentamicin resistance plasmids. A probe specific for the gentamicin resistance determinant hybridized to the plasmids of four isolates and to the chromosomes of two isolates. These findings suggest that clonal dissemination is not responsible for the spread of these resistant strains, that resistance determinants occur on different plasmids as well as on the chromosome of E. faecalis, and that the genetic determinants of resistance are related.     

In Vitro Evaluation of Tobramycin, a New Aminoglycoside Antibiotic

One hundred fifty-two strains of Escherichia coli, Klebsiella-Enterobacter, Pseudomonas aeruginosa, Proteus species, and Staphylococcus aureus were inhibited by 3.1 mug of tobramycin/ml in a broth-dilution method and showed zones of inhibition of 16 mm or more around a 10-mug tobramycin disc in the Kirby-Bauer method. Tobramycin was most active against S. aureus, 100% of strains being inhibited by 0.1 mug/ml. All strains of E. coli, K. pneumoniae, P. aeruginosa, and indole-positive Proteus species, and 80% of Enterobacter species were inhibited by 0.8 mug of tobramycin/ml, whereas only 48% of P. mirabilis strains were inhibited by this concentration. Tobramycin was approximately twice as active as gentamicin against S. aureus, four times as active against P. aeruginosa, slightly more active against E. coli and Enterobacter species, equally active against P. mirabilis, and slightly less active against K. pneumoniae. The minimal bactericidal concentrations of tobramycin and gentamicin were the same as or twice the minimal inhibitory concentrations for all strains except those of P. aeruginosa, against which greater concentrations of both gentamicin and tobramycin were required for bactericidal activity. Tobramycin sterilized cultures of S. aureus, E. coli, and P. aeruginosa, but the rate of bactericidal action was faster with a combination of tobramycin and carbenicillin than with either antibiotic alone in the same concentrations. Tobramycin retained potency in the presence of 200 to 600 mug of carbenicillin/ml for at least 6 hr of incubation at 37 C, but lost potency in the presence of 600 mug of carbenicillin/ml by 24 hr of incubation and in the presence of 800 mug/ml by 2 hr of incubation.     

Delayed resistance selection for doripenem when passaging Pseudomonas aeruginosa isolates with doripenem plus an aminoglycoside

Doripenem (formerly S-4661), a parenteral carbapenem, was tested in combination with an aminoglycoside (gentamicin) to determine the resistance selection of these codrugs during subinhibitory passaging using 6 Pseudomonas aeruginosa isolates. The organisms were selected based on doripenem and gentamicin MIC values to include isolates with MIC values near the susceptible breakpoints of both compounds and 1 strain highly resistant to gentamicin. Baseline MIC values were established for doripenem (2-8 microg/mL) and gentamicin (4 to >256 microg/mL) using reference broth microdilution methods, and passaging was carried out over 7 consecutive days. Doripenem MIC values increased 2 to >/=8-fold in 4 isolates, whereas 2 strains maintained the baseline doripenem MIC. When the experiment was performed with doripenem plus gentamicin, 3 strains maintained the original doripenem MIC values, 2 strains had a 2-fold increase, and only 1 strain showed a 4-fold increase in the doripenem MIC values. Previous studies have demonstrated doripenem to be more potent than other members in its class when tested against P. aeruginosa. The combination of doripenem and an aminoglycoside may be an effective treatment of infections caused by P. aeruginosa with elevated carbapenem MIC values with lower risk of selecting further resistance.     

Characterization and comparison of two penicillinase-producing strains of Streptococcus (Enterococcus) faecalis.

We identified two beta-lactamase-positive enterococci. One strain was high-level (MIC, greater than 2,000 microgram/ml) gentamicin resistant; the other was not (MIC, 12.5 microgram/ml). beta-Lactamase production was extrachromosomally mediated in both strains, and both strains showed an inoculum effect reversed by beta-lactamase inhibitors. The strain lacking high-level gentamicin resistance showed synergistic killing with a combination of penicillin, clavulanic acid, and gentamicin.     

Tobramycin: In Vitro Activity and Comparison with Kanamycin and Gentamicin

The in vitro activity of the aminoglycoside antibiotic tobramycin was demonstrated by broth dilution and single-disc methods on 50 isolates each of Staphylococcus aureus, Klebsiella or Enterobacter, indole-positive and -negative Proteus, Escherichia coli, and Pseudomonas aeruginosa. All organisms were inhibited by 6.25 mug or less of the drug/ml. Pseudomonas strains resistant to kanamycin or gentamicin or both were susceptible to tobramycin. Those strains which were inhibited by 6.25 mug of tobramycin/ml by the broth dilution method had zone diameters of 16 mm or more by the single-disc method. Of 313 organisms tested by the disc method, 3 strains were found to be resistant to tobramycin, 73 were resistant to kanamycin, and 18 were resistant to gentamicin. Tobramycin was found to have satisfactory in vitro activity against many clinically important organisms, including strains resistant to gentamicin and kanamycin.     

Role of mutations in DNA gyrase genes in ciprofloxacin resistance of Pseudomonas aeruginosa susceptible or resistant to imipenem.

In Pseudomonas aeruginosa, resistance to imipenem is mainly related to a lack of protein OprD and resistance to fluoroquinolones is mainly related to alterations in DNA gyrase. However, strains cross resistant to fluoroquinolones and imipenem have been selected in vitro and in vivo with fluoroquinolones. We investigated the mechanisms of resistance to fluoroquinolones in 30 clinical strains of P. aeruginosa resistant to ciprofloxacin (mean MIC, >8 micrograms/ml), 20 of which were also resistant to imipenem (mean MIC, >16 micrograms/ml). By immunoblotting, OprD levels were markedly decreased in all of the imipenem-resistant strains. Plasmids carrying the wild-type gyrA gene (pPAW207) or gyrB gene (pPBW801) of Escherichia coli were introduced into each strain by transformation. MICs of imipenem did not change after transformation, whereas those of ciprofloxacin and sparfloxacin dramatically decreased (25- to 70-fold) for all of the strains. For 28 of them (8 susceptible and 20 resistant to imipenem), complementation was obtained with pPAW207 but not with pPBW801. After complementation, the geometric mean MICs of ciprofloxacin and sparfloxacin (MICs of 0.3 microgram/ml and 0.5 microgram/ml, respectively) were as low as those for wild-type strains. Complementation was obtained only with pPBW801 for one strain and with pPAW207 and pPBW801 for one strain highly resistant to fluoroquinolones. These results demonstrate that in clinical practice, gyrA mutations are the major mechanism of resistance to fluoroquinolones even in the strains of P. aeruginosa resistant to imipenem and lacking OprD, concomitant resistance to these drugs being the result of the addition of at least two independent mechanisms.     

Plasmid-mediated gentamicin resistance of Pseudomonas aeruginosa and its lack of expression in Escherichia coli.

We isolated 11 nonconjugative plasmids mediating resistance to aminoglycoside antibiotics, including gentamicin, from Pseudomonas aeruginosa strains. Their genetic properties were investigated in both P. aeruginosa and Escherichia coli transformants. The plasmid molecular weights ranged from 11 x 10(6) to 24 x 10(6). A low level or complete absence of gentamicin resistance was observed when these plasmids were introduced into E. coli, but gentamicin resistance was restored when the plasmids were transferred back to P. aeruginosa from E. coli. Aminoglycoside-modifying enzyme activity was detected in P. aeruginosa harboring these plasmids, but was absent or greatly reduced in E. coli strains. This lack of expression may explain the observed decrease in aminoglycoside resistance.     

Properties of R plasmids determining gentamicin resistance by acetylation in Pseudomonas aeruginosa

Two clinical isolates of Pseudomonas aeruginosa, one a pyocin type 5 strain from Atlanta, could transfer gentamicin resistance by conjugation. Donor and recipient strains inactivated gentamicin by acetylation. The R plasmids, pMG1 and pMG2, also determined resistance to sisomicin, another substrate of gentamicin acetyltransferase I, sulfonamides, and streptomycin, but not resistance to kanamycin, neomycin, tobramycin, butirosin, or BB-K 8. They were transmissible to many strains of P. aeruginosa, including a Rec(-) strain, but not to Escherichia coli or other enterobacteriaceae. These R plasmids were compatible with R plasmids transmissible to P. aeruginosa from E. coli, including members of C, N, P, and W incompatibility groups. From a strain carrying pMG1 and a compatible plasmid, pMG1 was transferred independently but transfer of the second plasmid often resulted in cotransfer of pMG1. In contrast, pMG1 and pMG2 were incompatible with pseudomonas R plasmids R931 and R3108, and with R931 they readily formed recombinant plasmids. The four plasmids in this incompatibility group determine additional biological properties, including resistance to inorganic and organic mercury compounds, to ultraviolet light, and to certain deoxyribonucleic acid phages. pMG1 and pMG2 also phenotypically inhibited pyocin production. Consequently such R plasmids alter the phage and pyocin types of their host strains.