Thermosensitive Replication of a Kanamycin Resistance Factor

A strain of Proteus vulgaris isolated from the urinary tract of a patient with postoperative pyelonephritis and resistant to sulfonamide, streptomycin, tetracycline, and kanamycin (KM) was found to transfer only KM resistance by cell-to-cell conjugation. The genetic determinant controlling the transferable KM resistance was considered to be an R factor and was designated R (KM). Successive transfer of KM resistance was demonstrated also from Escherichia coli 20S0, which received the R (KM) factor, to other substrains of E. coli K-12 or Salmonella typhimurium LT-2. The transfer of the R (KM) factor was strongly affected by the temperature at which the mating culture was kept. The transfer frequency of R (KM) at 25 C was about 10(5) times higher than at 37 C. The R (KM) factor was spontaneously eliminated from the host bacterial cells when P. vulgaris was cultured at 42 C, but no elimination occurred at 25 C. This elimination of the R (KM) factor at elevated temperature was also observed when the R (KM) factor infected E. coli and S. typhimurium. On the other hand, a normal R factor could not be eliminated from the same E. coli host strain by cultivation at the higher temperature. We consider the thermosensitive transfer and the spontaneous elimination of the R (KM) factor at higher temperature to depend upon thermosensitive replication of the R (KM) factor.     

Episome-mediated Transfer of Drug Resistance in Enterobacteriaceae X. Restriction and Modification of Phages by fi R Factors

Watanabe, Tsutomu (Keio University, Tokyo, Japan), Toshiya Takano, Toshihiko Arai, Hiroshi Nishida, and Sachiko Sato. Episome-mediated transfer of drug resistance in Enterobacteriaceae. X. Restriction and modification of phages by fi(-) R factors. J. Bacteriol. 92:477-486. 1966.-An fi(-) R factor, which restricts phages lambda, T1, and T7 without modifying them, was found to restrict and not to modify an F(-)-specific phage, W-31, in Escherichia coli K-12, but not to restrict phage P-22 in Salmonella typhimurium LT-2, whereas other fi(-) R factors restricted and modified P-22 but not W-31; fi(+) R factors did not restrict these phages. Transduction and lysogenization with phages lambda and P-22 were reduced by these fi(-) R factors in K-12 and LT-2, respectively, and the transducing phages lambda and P-22 were modified by these fi(-) R factors. Spontaneous as well as ultraviolet-induced production of phage P-22 and zygotic induction of phage lambda were not significantly affected by any R factor. Injection of the nucleic acids of phages T1 and lambda was not affected by R factors, but the injected phage nucleic acids were rapidly broken down in the bacteria carrying fi(-) R factors. The nucleic acids of the modified phages were not broken down in these bacteria. It was assumed from these results that the mechanism of restriction of phages by fi(-) R factors is due to the breakdown of the injected phage nucleic acids by a deoxyribonuclease(s), presumably located near the cell surface in the cells carrying fi(-) R factors. The deoxyribonuclease(s), formed in the cells carrying the nonmodifying fi(-) R factor, is considered to be different from that synthesized in the cells carrying the modifying fi(-) R factors. It was further shown that the average burst sizes of the unmodified as well as modified phages are slightly reduced by the presence of the fi(-) R factors.     

R Factor-Mediated Resistance to Aminoglycoside Antibiotics in Pseudomonas aeruginosa

Conjugal transferability of drug resistance was examined, in eleven Pseudomonas aeruginosa strains which were isolated in Frankfurt. Four R factors were demonstrated from three strains using P. aeruginosa as recipients but they were nontransferable to Escherichia coli K12. Two R factors, i.e., R_ms146 and R_ms147, mediated resistances to tetracycline (TC), streptomycin (SM), sulfanilamide (SA), kanamycin (KM), lividomycin (LV), gentamicin C complex (GM) and 3′,4′-dideoxykanamycin B (DKB). They mediated the formation of aminoglycoside-inactivating enzymes, i.e., SM phosphotransferase, SM adenylyltransferase, KM and LV phosphotransferase 1, and GM and DKB 6′-N-acetyltransferase. TC resistance conferred by these R factors was due to impermeability of the drug. P. aeruginosa Ps 142 carried two kinds of R factor in one cell, R_ms148 (SM) and R_ms149 (SM·SA·GM·CPC) (CPC, carbenicillin). R_ms148 (SM) was transferable at a high frequency of 10^–1 and mediated the formation of SM phosphotransferase. R_ms149 mediated the formation of drug-inactivating enzymes, i.e., GM 3-N-acetyltransferase and β-lactamase, but did not inactivate SM. SM resistance was probably due to impermeability of the drug.     

Drug resistance and R plasmids in Escherichia coli strains isolated from broilers

In 1978, 1,021 Escherichia coli strains were isolated from 105 field broilers (F) and 1,058 strains from 106 broilers in a zootechnical experiment station (Z), and their drug-resistance patterns and the presence of conjugative R plasmids were compared. The resistance markers examined were tetracycline (TC), chloramphenicol (CM), streptomycin (SM), sulfonamides (SA), kanamycin (KM), and ampicillin (APC). The populations of individuals that excreted resistant strains were 100% in F and 58% in Z. Frequencies of isolation of drug-resistant strains among the total isolates were 93% in F and 36% in Z, indicating that the resistant strains are a rather high proportion of the intestinal flora in F but are slightly less prevalent in Z. The resistance pattern to (TC.SM.SA.KM) was seen at the highest frequency in both groups. Conjugative R plasmids were demonstrated more frequently in field broilers (F). The results reflect the wide use of antibiotics in the livestock industry, resulting in the appearance of drug-resistant strains mostly due to the presence of R plasmids.     

Drug Resistance of Enteric Bacteria

Drug resistance of 3,000 Shigella strains isolated in 1965 were investigated. These strains originated from 10 City Hospitals and 4 Prefectural Health Centers, which are located in different parts of Japan. One hundred and seventy strains which were resistant to 4 drugs, chloramphenicol (CM), tetracycline (TC), dihydrostreptomycin (SM), and sulfanilamide (SA), were selected at random from these stock cultures in this laboratory and the distribution of R factors in these isolates was examined. It was found that the strains all harbored R factors which were capable of transferring drug resistance by usual conjugal process. Among the strains carrying R factors, 85 per cent harbored a single type of R factor and 15 per cent carried two types of R factor in a cell. The latter is called the hetero-R state. Among the strains in the hetero-R state, isolation of strains harboring both R (SM.SA) and R (TC.CM.SM.SA) factors was most frequent. It was found that 25 R (SM.SA) factors isolated from strains in hetero-R had the genetic determinant iR^?, while most of the R (TC.CM.SM.SA) factors isolated from natural sources were iR⁺. When two types of R factor, R (SM.SA) and R (TC.CM.SM.SA) derived from the same host cells, were brought together in a host cell by superinfection with both factors, they were found to exist stably in a host bacterium. These results confirmed the stable existence of both factors in Shigella strains isolated from dysenteric patients.     

Gentic properties of an R factor carrying resistance to aminolgycoside antibiotics.

R factor Rms 151 is an fi+ R factor and belongs to a incompatibility group FII. It carries the genes governing resistance to various aminoglycoside antibiotics, i.e., kanamycin (KM), lividomycin (LV), gentamicin C complex (GM), and 3',4'-dideoxykanamycin B (DKB), in addition to those governing to tetracycline (TC), chloramphenicol (CM), sulfanilamide (SA), and ampicillin (APC). Electron microscopy observation disclosed that the Rms151 deoxyribonucleic acid was a circular form with length of 31.2 mum. A probable circular genetic map of Rms151 was proposed by genetic and biochemical studies, the genes being in the order of -tet-tra-amp-aad-sul-aph-cml-, in which aad and aph confer resistance to KM.GM.DKB by adenylytransferase or resistance to KM.LV by phosphotransferase, respectively.     

Drug resistance and conjugative R plasmids in fecal Escherichia coli strains isolated from healthy younger animals (chickens, piglets, calves) and children

A total of 11,777 Escherichia coli strains were isolated from 90 chickens, 103 piglets, 96 calves, and 104 children in 1979 in Gunma Prefecture and tested for drug resistance and the presence of conjugative R plasmids. The percentages of individuals that excreted drug-resistant strains were: chickens, 100%; piglets, 99%; calves, 100%; and children, 64%. The frequency of isolation of drug-resistant strains among the total isolates was: chickens, 98%; piglets, 93%; calves, 94%; and children, 41%. Frequency of isolation of R plasmids among the strains tested was: chickens, 48%; piglets, 33%; calves, 38%; and children, 10%. Resistance patterns of the strains isolated most frequently among the four groups were tetracycline (TC), sulfonamides (SA) in single resistance, TC.SA in double resistance, TC.streptomycin (SM).SA in triple resistance and TC.SM.SA. kanamycin (KM) in quadruple resistance. R plasmids were isolated frequently from animals (over 33%) but infrequently from children (about 10%). The high frequency of isolation of drug-resistant strains and R plasmids from animals was caused by the heavy use of chemicals in the period of growth of younger animals.     

Unstable Mutants of R Factor

Thirty mutants sensitive to tetracycline were obtained from an R100 factor capable of conferring resistance to tetracycline (TC), chloramphenicol (CM), streptomycin (SM) and sulfanilamide (SA). Among the TC sensitive mutants, three showed a high frequency of spontaneous loss from host strains. The genetic loci governing the stability of R factor in host bacteria were denoted as stb. The stb^– R factors have lost many of the properties of a wild type R factor, such as, the capability to sexually transfer drug resistance and host chromosome, to confer superinfection immunity and to inhibit F function. All of these properties did not revert to a wild type phenotype, suggesting that these mutations are deletions including genetic determinants governing both TC resistance and stability of R factor. Recombinational analysis between stb^– and stb⁺ R factors indicated that crossovers between the stb loci and those governing CM (or SM.SA) resistance took place at high frequency. No crossovers were detected between stb loci and those governing TC resistance, indicating that the stb loci are linked closely to the loci governing TC resistance.     

The Incidence of R Factors in Bordetella bronchiseptica Isolated from Pigs

Bordetella bronchiseptica strains isolated from the nasal cavities of young pigs in Japan from 1969 to 1972 were surveyed for drug resistance and distribution of R factors. Of 304 strains examined, 71 (23%) were resistant to either one or more of following three drugs, streptomycin (SM), sulfadimethoxine (SA), and aminobenzyl penicillin (APC). Triple (SM.SA.APC)-resistance was most frequent among these resistant strains. Strains of double (SM. SA)- or single (SM)- and (SA)-resistance were also isolated, but were very few in numbers. Of the 71 drug-resistant strains, 61 (86%) were found to carry R factors which were capable of conjugal transfer. All of these R factors had the triple (SM.SA.APC)-resistant markers and were identified as fi^– (no fertility inhibition) type. The (SM.SA.APC)-resistant strains carrying R factors had been isolated from pigs reared on various farms in different districts, and consequently the prevalence of B. bronchiseptica strains carrying R factors was considered to be relatively wide-spread in young pigs.     

Drug Resistance and Broad Geographical Distribution of Identical R Plasmids of Pasteurella piscicida Isolated from Cultured Yellowtail in Japan

An MIC test of 12 chemotherapeutic agents performed on 175 strains of Pasteurella piscicida collected from cultured yellowtail (Seriola quinqueradiata) in different areas of Japan from 1989 to 1991 revealed 152 strains (87%) with resistance to combinations of ampicillin (AP), chloramphenicol (CP), kanamycin (KM), nalidixic acid (NA), sulfamonomethoxine (SA), tetracycline (TC), and/or trimethoprim (TMP). The remaining 23 strains were sensitive to all the drugs tested: AP, cefazolin, CP, florfenicol (FF), furazolidone, KM, NA, novobiocin, SA, streptomycin, TC, and TMP. FF showed the most effective antibacterial activity against P. piscicida with MICs ranging from 0.004 to 0.6 μg/ml. One hundred and forty-nine of the 152 resistant strains carried transferable R plasmids encoding one of the Cp Km Sa Tc, Km Sa Tc, Km Sa, and Sa resistance. The most common resistance marker of transferable R plasmids identified in P. piscicida was Km Sa Tc. R plasmids encoding three different resistant markers were very similar on the basis of their digestion patterns with restriction endonucleases. There was homology among the DNAs of nine transferable R plasmids selected. Our findings suggest that multiple drug resistant strains of P. piscicida carrying transferable R plasmids with the same DNA structure are common in yellowtail farms and that the R plasmid has been retained within the P. piscicida population without change in their DNA structure according to geography and year.     

Genetic Stability of Various Resistance Factors in Escherichia coli and Salmonella typhimurium

Genetic stability of R factors was studied in Salmonella typhimurium LT-2 and Escherichia coli K-12. It was found that fi(+) R [or R(f)] factors were unstable in LT-2, losing their drug-resistance markers at high frequencies, and were stable in K-12; fi(-) R [or R(i)] factors were stable in both hosts. Both fi(+) and fi(-) R factors were genetically stable also in recombination-deficient mutants of K-12. An fi(+) R factor, which was unstable in S. typhimurium LT-2 wild type, was relatively stable in a recombination-deficient mutant of LT-2. In the spontaneous loss of the drug-resistance markers of fi(+) R factors in LT-2, the markers for sulfanilamide, streptomycin, and chloramphenicol resistance were lost together at high frequencies and the tetracycline marker was retained stably. The remaining drug-resistance markers of the spontaneous segregants of LT-2 were transmissible to K-12 by mixed cultivation, indicating that they were still in the form of R factors.     

Episome-mediated transfer of drug resistance in Enterobacteriaceae. IX. Recombination of an R factor with F

Watanabe, Tsutomu (Keio University, Tokyo, Japan), and Chizuko Ogata. Episome-mediated transfer of drug resistance in Enterobacteriaceae. IX. Recombination of an R factor with F. J. Bacteriol. 91:43-50. 1966.-R factors can be transduced in Salmonella typhimurium with phage P-22, and a majority of the drug-resistant transductants are unable to transfer their drug resistance by cell-to-cell contact, as we have previously reported. Several exceptional types of transductants of S. typhimurium, with the markers of resistance to sulfonamide, streptomycin, and chloramphenicol, were recently obtained by transduction with phage P-22 of a four-drug-resistance R factor carrying the markers of resistance to sulfonamide, streptomycin, chloramphenicol, and tetracycline. They were exceptional in that they had low conjugal transferability of their drug resistance. When one of these exceptional transductants (38R) was transferred to an F(+) strain of Escherichia coli K-12, 38R acquired high transferability in its further transfer. This high transferability was found to be due to the recombination of 38R with F. Transductant 38R was of the fi(+) (fi = fertility inhibition) type, and did not show superinfection immunity against fi(+) and fi(-) R factors. The recombinant 38R.F was genetically very stable and resistant to elimination with acridines. It did not show superinfection immunity against fi(+) and fi(-) R factors, but did show superinfection immunity against F. Further, 38R.F did not restrict a female-specific phage (W-31), unlike wild-type F. F(-) and R(-) segregants were isolated from this recombinant 38R.F, and these segregants exhibited genetic characteristics different from the original R, its transductant 38R, and wild-type F.     

Comparisons of F Factors and R Factors: Existence of Independent Regulation Groups in F Factors

The similarity of sex pili mediated by F factors and R(fi(+)) factors and the ability of R(fi(+)) factors to control by repression the functioning of pilus genes encoded by the F factor suggested that F factors and R(fi(+)) factors are closely related. Further comparisons of the episomal properties of F factors and R(fi(+)) factors, however, indicated many differences. F factors contain information for a restriction system for phages phiII and T7. Cells containing R factors are sensitive to these phages. Furthermore, R(fi(+)) factors do not repress the F factor phiII restriction system in cells containing both an R(fi(+)) factor and an F factor. R factors and F factors are heteroimmune episomes. In addition, an R(fi(+)) factor in cells containing both an R factor and an F factor does not fully repress the expression of F-factor immunity to an incoming second F factor. R-factor and F-factor replication systems are not identical. Wild-type F-factor replication genes will complement the mutant F(ts114)lac(+) replication genes in cells containing two F factors. The F(ts114)lac(+) episome is retained when these cells are grown at 42 C; however, cells containing an R(fi(+)) factor and F(ts114)lac(+) lose the F(ts114)lac(+) when grown at 42 C, at the same rate as cells containing only the F(ts114)lac(+). The replication system of the R(fi(+)) factor will not complement the mutant F(ts114)lac(+) replication system.     

Studies of Drug Resistance and R Factors in Bacteria from Pond-Cultured Salmonids

Considerable fractions of gram-negative bacilli, Aeromonas liquefaciens, A. salmonicida, Pseudomonas, Hafnia and unidentified Enterobacteriaceae, isolated from the intestinal tracts of cultured Amago (Oncorhynchus rhodurus macrostomus) from 23 Amago-ponds and of Yamame (O. masou ishikawae) from one pond as well as the water of 3 ponds in Shiga, Gifu and Nagano Prefectures and Tokyo Metropolis in Japan were found to be multiple-drug-resistant. Of these drug-resistant strains, A. salmonicida and A. liquefaciens carried R factors at high frequencies and to be prevalent in many Amago ponds throughout Japan. These R factors had markers of resistance to SA, SA.TC, SA.SM.CM. All the R factors belonged to fi^– type.     

Drug resistance of enteric bacteria. VII. Recombination of R factors with tetracycline-sensitive mutants

Hashimoto, Hajime (Gunma University, Maebashi, Japan), and Susumu Mitsuhashi. Drug resistance of enteric bacteria. VII. Recombination of R factors with tetracycline-sensitive mutants. J. Bacteriol. 92:1351-1356. 1966.-The transmissible drug-resistance factor R is able to confer resistance to tetracycline (TC), chloramphenicol (CM), streptomycin (SM), and sulfonamide (SA) on a host bacterium when infected by cell-to-cell contact. Tetracycline-sensitive mutants were isolated from either CM- or SM-sensitive mutants of an R factor. Among 30 mutants isolated, 10 were point mutants which could recombine with each other, forming recombinant R factors able to grow on plates containing 50 mug/ml of TC. The recombination frequency of TC-resistant recombinants was 10(-2) to 10(-3) in bacterial cells carrying two types of TC-sensitive R factors by superinfection with both factors. Segregational patterns of the various markers on the R factor, i.e., chl, str, sul, and m, the locus determining R mating, and their linkage order, were investigated among TC-resistant recombinants of the R factor. When TC was used as the selective drug, the tet locus mapped on the R factor as an end marker. In view of the fact that these results are inconsistent with the linkage order of various markers reported previously, a circular genetic structure for the R factor which includes five tet-s and three chl-s loci is presented.     

Integration of temperature-sensitive nonconjugative plasmid carrying kanamycin gene into conjugative R plasmids.

A nonconjugative R plasmid, rMS3, whose molecular weight was 2.4 X 10(7) daltons, possessed a kanamycin resistance gene and was thermosensitive in its maintenance in Escherichia coli strains. We mobilized rMS3 with a conjugative R plasmid, R100 or T-tet, and obtained cointegrates carrying all the parental resistance markers. Various markers of the cointegrates were frequently deleted by P1 transduction and the deletion patterns among the different cointegrates were differed from each other. The cointegrates were thermoresistant, but the thermosensitive replicon could be segregated from the thermoresistant cointegrate by deletion. Some cointegrates between rMS3 and T-tet showed a derepressed state of transferability because of the integration of rMS3 and T-tet showed a derepressed state of transferability because of the integration of rMS3 into the regulator gene of the transfer loci. The genome size of the cointegrate so far tested was the sum of the sizes of the parental plasmids, indicating that the whole genome of rMS3 could integrate into various sites of the conjugative plasmids R100 and T-tet.     

Genetic and Molecular Properties of an Infectious Antibiotic Resistance (R) Factor Isolated from Klebsiella

A Klebsiella strain of human origin that was resistant to ampicillin, chloramphenicol, kanamycin, neomycin, streptomycin, and tetracycline was found to have all of these resistances associated with a R factor and a satellite molecular species of deoxyribonucleic acid (DNA) with an average buoyant density of 1.710 in cesium chloride gradients. There was no evidence of the existence of DNA with other buoyant densities. The strain bears two separable mutations for chloramphenicol resistance, both of which are associated with the R factor (KR9). Exposure of the Klebsiella strain to acridine derivatives or to ethidium bromide (which was more efficient) resulted in partial losses of resistance accompanied by the disappearance of the satellite DNA peak or shifts in its density. The R factor and its component genes were conjugally transmitted across generic boundaries and maintained in new hosts with different efficiencies. The basis of this difference lies not only in the efficiency of conjugal transfer but also in the stability of the components after transfer. All of the resistance genes and the resistance transfer factor were cotransducible by phage Plkc from Escherichia coli. Partially resistant strains could be reconstituted to full resistance or to a recombined pattern of partial resistance by conjugation with donors having complementary resistance patterns. This recombination serves as an efficient mechanism for rescuing superinfecting genes that are otherwise intracellularly excluded. KR9 is an fi⁺ type of R factor which in the natural state does not appear to be as repressed in conjugal transfer as other R factors.     

Drug resistance among Mycobacterium tuberculosis strains isolated in Taiwan during 1975.

455 strains of Mycobacterium tuberculosis were isolated from patients with history of treatment in Taiwan Provincial Tuberculosis Control Bureau and tested for resistance against various antituberculosis agents including streptomycin (SM), paraaminosalicylic acid (PAS), isoniazid (INH), cycloserine (CS), prothionamide (1321TH), kanamycin (KM), ethambutol (EMB), and rifampicin (RFP). In vitro resistance to SM and INH was more frequently found than others and the resistance to a single drug was more common than multiple resistance.     

Drug Resistance of Staphylococci VII. Genetic Determinants Responsible for the Resistance to Tetracycline, Streptomycin, Sulfanilamide, and Penicillin

A genetic analysis of resistance to tetracycline (TC), streptomycin (SM), sulfanilamide (SA), and penicillin G was carried out through transduction with phage lysates obtained from a multiply resistant strain of Staphylococcus aureus by ultraviolet irradiation. All transductants acquired resistance to both TC and SA, even when singly selected for either SA or TC resistance. The locus responsible for TC resistance could not be separated genetically from that for SA resistance. On the other hand, in transduction of SM resistance, about 30% of the transductants jointly acquired resistance to both TC and SA. These observations suggest that the loci governing resistance to TC, SA, and SM exist close together on a single genetic unit, this probably being the chromosome.     

Frequency of R Factor-mediated Multiple Drug Resistance in Klebsiella and Aerobacter

A comparative study was done on the transfer frequency of R factors from 90 strains of multiple drug-resistant Aerobacter and 81 strains of Klebsiella to Escherichia coli CSH-2 (F(-), met(-), pro(-), Nal-r). The most common resistance patterns for the Aerobacter isolants were ampicillin streptomycin chloramphenicol tetracycline and ampicillin streptomycin chloramphenicol tetracycline kanamycin neomycin; for the Klebsiella isolants, the most common resistance pattern was ampicillin kanamycin streptomycin tetracycline chloramphenicol neomycin. R factors were isolated from 14.1% of the Aerobacter strains; 61.5% of these R factors harbored R determinants for ampicillin streptomycin tetracycline. R factors were isolated from 79.1% of the Klebsiella strains; four R factors were isolated with significant frequency; streptomycin chloramphenicol kanamycin neomycin, 37.5%; ampicillin streptomycin tetracycline kanamycin neomycin, 14.1%; ampicillin streptomycin tetracycline, 12.5%; and streptomycin chloramphenicol tetracycline, 12.5%.Chloramphenicol, kanamycin, and neomycin resistance was rarely transferred from the Aerobacter strains, although over 50% of the clinical isolants possessed resistance to these antibiotics. In contrast, over 75% of the Klebsiella strains transferred resistance to chloramphenicol, kanamycin, neomycin. Highest frequency of transferred resistance to individual drugs in the Aerobacter strains was to streptomycin (14.8%), whereas in the Klebsiella group resistance to four drugs was transferred at a very high frequency: streptomycin (80.8%), chloramphenicol (78.5%), kanamycin (76.4%), and neomycin (75.9%).