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Chloramphenicol acetyltransferases specified by fi minus R factors.

Chloramphenicol acetyltransferases specified by fi(-) (F fertility-noninhibiting) R factors were compared with those specified by the fi(+) R factors R1, 222, and R6-S. Three classes of enzyme were distinguished: class I was determined by group N fi(-) R factors and was indistinguishable from the enzyme determined by fi(+) R factors, class II was correlated with group W fi(-) R factors, and class III was determined by R387, an unclassified fi(-) R factor.

Acetyltransferases↗

Two enzymes which phosphorylate neomycin and kanamycin in Escherichia coli strains carrying R factors.

Two enzymes which phosphorylate neomycin and kanamycin have been detected in Escherichia coli strains carrying R factors. Neomycin-kanamycin phosphotransferase I can phosphorylate neomycin and kanamycin, but not butirosin. Neomycin-kanamycin phosphotransferase II can phosphorylate all three antibiotics. The enzymes also differ in their response to certain inhibitors of phosphorylation. Neomycin-kanamycin phosphotransferase II is found in certain strains of Pseudomonas aeruginosa.

Drug Resistance, Microbial↗

Molecular characterization of the R factors implicated in the carbenicillin resistance of a sequence of Pseudomonas aeruginosa strains isolated from burns.

An outbreak of R-factor-mediated carbenicillin resistance in Pseudomonas aeruginosa in burned patients in March 1969 was followed by a second outbreak 6 months later. No R-factor-carrying P. aeruginosa strains were detected in the intervening period but R-factor-determined lactamase was commonly encountered, particularly in Klebsiella aerogenes strains. A comparison of the molecular properties of the R factors in pseudomonads from the first and second phases with those in the Klebsiella strains from the intervening period showed them to be very closely related. A single R-factor type therefore may have been maintained in the Burns Unit between the two Pseudomonas outbreaks as a plasmid conferring resistance to ampicillin in K. aerogenes.

Burns↗

Mutation of R factors capable of specifying hypersynthesis of penicillinase.

When strains harboring R factor and showing ampicillin (APC) resistance were inoculated on plates containing various concentrations of APC, mutants carrying various levels of APC resistance occurred at high frequencies. Increases in the level of APC resistance were due to a quantitative increase in the formation of penicillinase. By conjugation experiments and transduction analysis, the mutation was found to affect the gene (amp) governing APC resistance on the R factor. The R-factor mutants carrying high and stable APC resistance were conjugally transferred at the same frequency as their parent R factors, and the level of their resistance to drugs other than APC was not distinct from that conferred by their parents. Such R-factor mutants could easily be obtained from wild-type R factors carrying low APC resistance. The hypersynthesis of penicillinase by such R-factor mutants was considered to be due to the replication of the amp gene on the R factor at hyper-rates and the integration of multiple copies of the amp gene (amp-hyper) in the R-factor genome.

Ampicillin↗

Relationships between curing of the F episome by rifampin and by acridine orange in Escherichia coli.

Subinhibitory doses of rifampin cured F(+)Escherichia coli cells from the episome. The target of the drug was transcription because E. coli mutants with a ribonucleic acid polymerase resistant to rifampin were not cured. The experimental conditions required for optimal curing with rifampin very closely resembled those required for curing with acridine orange. Mutants were found which are more resistant to curing by both acridine orange and rifampin. Probably the two drugs affect a common metabolic step, or alternatively they may inhibit the synthesis of a factor which is necessary for the replication of the episome.

Acridines↗

Interbacterial transfer of R factor in humans.

In vivo interbacterial transfer of R factor was observed in two patients. Resistance to ampicillin, tetracycline, streptomycin, and chloramphenicol was transferred from Escherichia coli in the intestine of a patient with shigellosis to a Shigella sonnei strain. In a second patient with partial ureteral obstruction, a resistant E. coli strain transferred ampicillin, tetracycline, and streptomycin resistance to a Proteus mirabilis strain.

Adolescent↗

Demonstration of R factors in Bordetella bronchiseptica isolated from pigs.

Transferability of drug resistance in strains of Bordetella bronchiseptica isolated from pigs was examined. These strains were resistant to sulfadimethoxine (SA, more than 1,600 mug/ml), streptomycin (SM, more than 800 mug/ml), and aminobenzyl penicillin (APC, 200 mug/ml). All of them could transfer their drug resistance as one unit to a sensitive strain of Escherichia coli as well as to B. bronchiseptica by mixed cultivation. The transferred SM-SA-APC resistance in the exconjugants of E. coli and B. bronchiseptica was also transmissible by mixed cultivation. But the transfer of triple resistance was not mediated by the cell-free filtrate of the drug-resistant strain of B. bronchiseptica which was used as donor. The triple resistance in the exconjugant E. coli ML1410 was found to be eliminated as a whole by treatment with acriflavin. According to these results, it could be safely concluded that R factors carrying SM-SA-APC resistance were demonstrated in strains of B. bronchiseptica isolated from pigs.

Animals↗

Sex pili mutants isolated by macarbomycin treatment.

Macarbomycin (MM) preferentially kills Escherichia coli carrying episomes derepressed for sex pili formation (drd episomes). Using MM, we have isolated various types of mutants from both F-lac.tet and R100-1 episomes and have classified them into two types by sex pili formation. Among 26 mutants, 20 could not produce pili and had lost both the ability for conjugal transmission and sensitivity to male phages. From 12 of these 20 mutants, revertants capable of pili formation could be obtained. Most of these had simultaneously regained susceptibility to MM, indicating that the MM susceptibility of E. coli carrying drd episomes is due to pili formation. Three of the revertants could not confer MM susceptibility on their host and two of them could not produce normal pili, indicating that there are some types of pili insensitive to MM. The six MM-resistant mutants could produce pili, but the frequency of pili formation was slightly lower than that of their parent episomes. Mutants of this type exhibited various degrees of decrease in conjugal transferability or male phage sensitivity, and one mutant produced pili that were altered in shape. It is suggested that this latter mutant became MM-resistant as a result of changes in the chemical composition of its pili.

Anti-Bacterial Agents↗

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.

Anti-Bacterial Agents↗

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.

Carbon Radioisotopes↗

Curing of an R factor Escherichia coli by trimethoprim.

R factor 1818, which we have shown previously to be eliminated by thymine starvation, was cured from three strains of Escherichia coli K-12 by overnight exposure to trimethoprim. Elimination was abolished in the presence of added thymine or thymidine, which suggests that curing is the result of the induction of thymineless conditions by trimethoprim. Starvation of the required amino acids proline and histidine had little effect on elimination, whereas methionine deprivation enhanced it. R factor curing was abolished by the presence of chloramphenicol, and it is concluded that protein synthesis is required for elimination to occur. It is suggested that elimination may result from the activity of a nuclease which is synthesized or induced during both direct thymine starvation and by trimethoprim treatment.

Amino Acids↗

Aminoglycoside uptake increased by tet gene expression.

The expression of extrachromosomal tet genes not only confers tetracycline resistance but also increases the susceptibilities of gram-negative bacteria to commonly used aminoglycoside antibiotics. We investigated the possibility that tet expression increases aminoglycoside susceptibility by increasing bacterial uptake of aminoglycoside. Studies of [3H]gentamicin uptake in paired sets of Escherichia coli HB101 and Salmonella typhimurium LT2 expressing and not expressing tet showed that tet expression accelerates energy-dependent [3H]gentamicin uptake. Increased [3H]gentamicin uptake was accompanied by decreased bacterial protein synthesis and bacterial growth. Increased aminoglycoside uptake occurred whether tet expression was constitutive or induced, whether the tet gene was class B or C, and whether the tet gene was plasmid borne or integrated into the bacterial chromosome. tet expression produced no measurable change in membrane potential, suggesting that tet expression increases aminoglycoside uptake either by increasing the availability of specific carriers or by lowering the minimum membrane potential that is necessary for uptake.

Anti-Bacterial Agents↗

Characterization and extrachromosomal control of bacteriocin production in Staphylococcus aureus.

About 6% of the tested strains of Staphylococcus aureus produced antagonistic substances against staphylococcal indicator strains. The production was low in liquid cultures and could not be induced by ultraviolet irradiation or by treatment with mitomycin C. The antagonistic substances could be classified into at least five groups on the basis of their properties and cross-resistance pattern. One group consisted of lytic enzymes and the four others of staphylococcins. One of the four types of staphylococcins was active against S. aureus only, and the three other types had a broader inhibitory spectrum against gram-positive organisms but not against gram-negative bacteria. A relationship was found between some groups of producing strains and their phage type. The ability to produce staphylococcins was eliminated spontaneously upon storage and more rapidly by treatment with ethidium bromide, acriflavine, acridine orange, sodium dodecyl sulfate, and growth at 42 C. The resistance to several inorganic salts was co-eliminated. No co-elimination of penicillinase production was observed. Selective effects during elimination were ruled out, and the results suggest that the genes for staphylococcin production are plasmid-borne determinants.

Bacteriocins↗

Transmissible resistance to penicillin G, neomycin, and chloramphenicol in Rhizobium japonicum.

The genetic basis for resistance to a number of antibiotics was examined in Rhizobium japonicum. Resistance to penicillin G, neomycin, and chloramphenicol appears to be mediated by an extrachromosomal element similar to that found in the Enterobacteriaceae. Resistance to these antibiotics was eliminated from cells by treatment with acridine orange, and resistance to all three antibiotics could be transferred en bloc to Agrobacterium tumefaciens under conditions excluding transformation or transduction as possible genetic mechanisms.

Acridines↗

Transferable antibiotic resistance in enteropathogenic Escherichia coli between 1948 and 1968.

Enteropathogenic strains of Escherichia coli which had been isolated in the United Kingdom during three periods between 1948 and 1968, namely 1948 to 1951, 1957 to 1960, and 1967 to 1968, were tested for susceptibility to ampicillin, streptomycin, tetracycline, chloramphenicol, and sulphonamides. Antibiotic-resistant strains were tested for their ability to transfer antibiotic resistance to an antibiotic-susceptible strain of E. coli K-12. A relatively high proportion of strains isolated between 1948 and 1951 was resistant to ampicillin, streptomycin, or sulphonamides. None of these strains transferred ampicillin or streptomycin resistance, but sulphonamide resistance was R-factor-mediated in three out of 14 sulphonamide-resistant strains. Resistance to tetracycline and chloramphenicol was rare before 1951 but had become common among enteropathogenic E. coli by 1957. Much of the antibiotic resistance of bacteria isolated between 1957 and 1960 was R-factor-mediated, and transferable resistance was about as prevalent among E. coli isolated between 1957 and 1960 as among strains isolated in 1967 and 1968. Nevertheless, there was no appreciable increase in the overall incidence of antibiotic resistance among these enteropathogenic strains of E. coli between 1957 and 1968, although transferable antibiotic resistance was common during this period. These results do not suggest that the emergence of transferable antibiotic resistance will inevitably lead to the rapid development of antibiotic resistance among this group of bacteria.

Anti-Bacterial Agents↗

Relationship between chloramphenicol acetyltransferase activity and the number of resistance genes.

Various mutants of an Escherichia coli K-12 strain were prepared, in which a chloramphenicol (CM) resistance gene (cml) derived from an R factor, R100-1, was integrated into the chromosome. The CM acetyltransferase (CATase) activity of these strains and the strain carrying R100-1 were determined during exponential growth with the following results. (i) The CATase activity varied, depending upon the site of integration of the cml gene on the chromosome. Activity was found to be higher when the cml gene was integrated nearer the replication origin of the chromosome, the total enzyme activity found was the sum of activities coded by each gene separately. (iii) When the cml gene was in a cytoplasmic state on an R factor, R100-1, the expressed enzyme activity was four-to eightfold higher than that in the chromosomal state, suggesting the existence of about four to eight copies of R factor per chromosome. The CATase activity returned to the level of that expressed by R100-1 when the chromosomal cml gene was detached and picked up by an R factor.

Acetyltransferases↗

Antimicrobial resistance and R factors in Salmonella isolated in California (1971-1972).

The antimicrobial resistance of 2,246 strains of Salmonella isolated from humans in California was determined. Resistance to one or more of the 12 antimicrobial agents tested was found in 32% of the isolates. Salmonella typhimurium strains represented 31% of the serotypes isolated; 49% of these strains were resistant. Fifty-one percent of S. heidelberg, 42.5% of S. newport, and 40% of S. saint paul strains were resistant. Seventy-seven percent of all resistant serotypes were resistant to two or more of the antimicrobial agents tested. R factors were demonstrated in 70% of the multiply resistant strains. Resistance of the Salmonella strains to one or more of the 12 antimicrobials tested and the frequency of resistance to tetracycline and ampicillin were significantly greater than resistance reported in other studies performed in this country. Chloramphenicol resistance was encountered in 33 (1.5%) of the Salmonella isolates; 19 of these were S. typhi demonstrating a pattern of resistance to streptomycin, sulfisoxazole, tetracycline, and chloramphenicol and carrying an R factor capable of transferring the complete pattern of resistance.

California↗

R factor-mediated and chromosomal resistance to ampicillin in Escherichia coli.

Sixty-four ampicillin-resistant strains of Escherichia coli were studied. Six characters were examined: (i) resistance to ampicillin, cephalothin, and carbenicillin, (ii) synergy between ampicillin and cloxacillin, (iii) level of beta-lactamase activity after osmotic shock, (iv) transferability of ampicillin resistance, (v) immunological characterization of the enzyme, and (vi) determination of substrate profiles. One class of strains was found in which synthesis of beta-lactamase is inferred to be plasmid mediated; these strains are highly resistant to ampicillin and carbenicillin, sensitive to cephalothin, do not show synergism between ampicillin and cloxacillin, and reveal a high enzymatic activity after osmotic shock. A second class is formed by strains for which beta-lactamase synthesis is inferred to be chromosomal; these strains present a low resistance level to ampicillin, are sensitive to carbenicillin and resistant to cephalothin, show a synergism between ampicillin and cloxacillin, and reveal a very low enzymatic activity after osmotic shock. These characters may be used to differentiate periplasmic and cell-bound beta-lactamases.

Ampicillin↗