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.     

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.     

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.     

Detection of R+ Bacteria in Cultured Marine Fish,Yellowtails (Seriola quinqueradiata)

Many drug-resistant gram-negative bacilli were isolated from the intestinal tracts of yellowtails (Seriola quinqueradiata) cultured on farms in various parts of Kochi and Ehime Prefectures, Shikoku Island. They were Vibrio, Pseudomonas, Proteus, Citrobacter and some unidentified Enterobacteriaceae. Of these drug-resistant strains, Vibrio and Pseudomonas were found to carry R factors in high frequencies. These R factors had four types of resistance markers, SA, SA. CM, SM. CM. ABP. and SA. SM. CM. TC. All R factors were found to belong to the fi^– type. In contrast, only one drug-resistant gram-negative bacillus was detected in a cultured yellowtail on farms near Numazu City, Shizuoka Prefecture.     

Integration of Chloramphenicol Resistance Gene of an R Factor on Escherichia coli Chromosome

An unstable mutant R factor conferring only chloramphenicol (CM) resistance was obtained by spontaneous segregation. After storage in broth culture, a stable CM-resistant mutant was obtained and its CM-resistance could not be cured by treatment with acriflavine or transduced to a recombination-deficient strain of Escherichia coli K12. Recombinational analysis indicated that the cml gene governing CM resistance had been integrated into the E. coli chromosome and closely linked with met B locus. The cml gene was co-transduced with both met and arg markers by phage P1, and the linkage order was considered to be mtl-cml-met-arg-thi. When the strain carrying this chromosomal CM-resistance was infected with a transferable R (TC) factor capable of conferring tetracycline (TC) resistance, the CM-resistance became transferable by conjugation. This mechanism is considered to account for the formation of the recombinant R (TC.CM) factor.     

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 of Enteric Bacteria

A nontransferable R₂₁ (TC) factor was obtained by transduction of R₁₀ (TC.CM.SM.SA) with phage epsilon in group E Salmonella. The R₂₁ (TC) factor acquired transmissibility by the normal conjugal process when group E Salmonella strains harboring R₂₁ (TC) factor were infected with wild-type F or R₁₆ (CM) factor. This transmissibility at high frequency was accounted for by the formation of the recombinant F TC and R₁₀ (CM) TC factors. The F TC and R₁₆ (CM) TC factors were genetically the same as the original F and R₁₆ (CM) factors, except for the ability to confer TC resistance. In the transduction of F TC factor with phage P1, a dF TC (d: defective) factor was obtained that was defective in many F properties, such as the ability to introduce host chromosome and produce male substance, but was capable of transducing TC resistance (dF TC-infection) at low frequency.     

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 VIII. Chromosomal Location of Nontransferable R Factor in Escherichia coli

The R₂₁(TC) factor, obtained by transduction of the R₁₀(TC.CM.SM.SA) factor with phage ε to group E Salmonella, is not transferable by the normal conjugal process. However, when R₂₁(TC)⁺ transductants are infected with the F₁₃ factor, the nontransferable R₂₁(TC) factor acquires transmissibility by conjugation. R₂₁(TC)⁺ conjugants of Escherichia coli K-12, to which only the R₂₁(TC) factor was transmitted by cell-to-cell contact from an F′ R⁺ donor, were still unable to transfer their R₂₁(TC) factor by conjugation. In crosses between Hfr and F⁻E. coli K-12 strains containing R₂₁(TC), the gene responsible for tetracycline resistance was located on the E. coli K-12 chromosome between lac and pro, near lac.     

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.     

EcoRI restriction endonuclease map of the composite R plasmid NR1.

A physical map of the composite R plasmid NR1 has been constructed using specific cleavage of deoxyribonucleic acid (DNA) by the restriction endonuclease EcoR-. Digestion of composite NR1 DNA by EcoRI yields thirteen fragments. The six largest fragments (designated A to F) are from the resistance transfer factor component that harbors the tetracycline resistance genes (RTF-TC). The seven smallest fragments (designated G to M) are from the r-determinants component that harbors the chloramphenicol (CM), streptomycin-spectinomycin (SM/SP), and sulfonamide (SA) resistance genes. The largest fragment of several RTF-TC segregants of NR1 that have deleted the r-determinants component is 0.8 X 10(6) daltons larger than fragment A of composite NR1. Only a part of fragment H of the r-determinants component is amplified in transitioned NR1 DNA in Proteus mirabilis, which consists of multiple, tandem sequences of r-determinants attached to a single copy of the RTF-TC component. Both of these changes can be explained by the locations of the excision sites at the RTF-TC: r-determinants junctions that are involved in the dissociation and reassociation of the RTF-TC and r-determinants components. The thirteen fragments of composite NR1 DNA produced by EcoRI have been ordered using partial digestion techniques. The order of the fragments is: A-D-C-E-F-B-H-I-L-K-G-M-J. The approximate locations of the TC, CM, SM/SP, and SA resistance genes on the EcoRI map were determined by analyzing several deletion mutants of NR1.     

Mapping andfi character determination of R factors ofEnterobacter cloacae DF13

In conjugation experiments betweenEnterobacter cloacae DF13 andEscherichia coli K12, resistances against tetracycline, sulfanilamide, streptomycin, and chloramphenicol were nearly always transferred simultaneously. These properties could be transferred fromE. coli exconjugants by transduction to a drug-sensitiveE. coli K12 strain with bacteriophage P1kc. It may be inferred thatEnt. cloacae DF 13 harbours a multiple R factor, which promotes its own transfer. This R factor was found to be of thefi ⁺ type. The molecular nature of this R factor was studied by labelling the DNA of an exconjugant with³H-thymidine, careful lysis, sedimentation of the chromosomal DNA, and characterization of the circular DNA by sucrose-gradient centrifugation, equilibriumdensity centrifugation in CsCl containing ethidium bromide and by electron microscopy. By these methods the multiple R factor was identified as a circular DNA molecule with a contour length of 22.6 Μm, corresponding to a molecular weight of 45 × 10⁶ daltons. A segregant R factor harbouring resistance against tetracycline only, was found to have a contour length of 16.0 Μm and a sedimentation constant of 58 S. In addition to the multiple R factor, the wild-type strain harboured a plasmid with a sedimentation constant of 38 S, corresponding to a molecular weight of 16 × 10⁶ daltons. The function of this plasmid is unknown. After many transfers on agar slants spontaneous segregation of the R factor was observed and several types of segregants were obtained. In most segregants, resistance against streptomycin could not be transferred by conjugation and could not be mobilized by other sex factors. Some of these segregants had acquired a requirement for methionine; in these, the streptomycin-resistance determinant may be integrated into the chromosome. The resistance pattern of the various types of segregants and exconjugants allowed to draw a circular map of the R factor. The order of markers is ---tet---rtf---sul---str---cml-. After short-term conjugation experiments most exconjugants were found to have received resistance against sulfanilamides only. This resistance determinant does not promote its own transfer by conjugation but could be mobilized by other sex factors. An exconjugant become resistant against tetracycline and sulfanilamide, was found to harbour two independent plasmids of which only that carrying resistance against tetracycline promoted its own transfer. Consequently a second R factor, determining resistance against sulfanilamide alone must be present inEnt. cloacae DF13. This R factor was identified as a circular DNA molecule with a sedimentation constant of 26 S, a contour length of 2.6 Μm and a buoyant density of 1.709. From a strain harbouring the independent R(SA) plasmid and an R(TC) fragment of the multiple R factor, transductants resistant against sulfanilamide were obtained. These were found to harbour an R(SA) plasmid with properties of a defective Rfi ⁺ transfer factor. Most probably these plasmids resulted from recombination between the R(SA) plasmid and the Rtf region of the R(TC) fragment. The author published previously under the name of “G. A. Tieze”. The technical assistance of Miss J.T.M.P.A. Havermans, Mrs. A. Mak-Zuidervaart, and Mr. M. V. M. Lafleur is gratefully acknowledged. The authors thank Dr. E. F. J. van Bruggen and Dr. D. Ellens for the electronmicroscopical measurements.     

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.     

Transition of the R factor NR1 and Proteus mirabilis: level of drug resistance of nontransitioned and transitioned cells.

When Proteus mirabilis harboring the R factor NR1 is cultured in Penassay broth containing 100 mug of chloramphenicol (CM) per ml, there is an amplification in the number of copies of the r-determinants per cell. Under these conditions, R factors harboring multiple tandem sequences of r-determinants are formed. Autonomous poly-f-determinants consisting of multiple copies of r-determinants are also formed. This phenomenon has been referred to as the "transition". Transitioned cells have considerably higher levels of resistance to CM and streptomycin (SM), but not to tetracycline (TC), than do nontransitioned cells and grow more rapidly in medium containing either CM or SM. There is essentially no difference in growth rates between transitioned and nontransitioned cells in drug-free medium. The higher level of resistance of transitioned cells to SM has made it possible to investigate the mechanism of the transition. Using replica plating, it has been possible to isolate spontaneously occurring transitioned cells from a nontransitioned population which appear to outgrow the nontransitioned cells during growth in medium containing 100 mug of CM per ml. If transiitoned cells are subsequently cultured in drug-free medium, the cells return gradually to the nontransitioned state, which has been referred to as the "back-transition was monitored by examining the level of resisitance of the cells to SM. In both situations the cell populations were found to be heterogeneous, consisting of a mixture of nontransitioned and transitioned cells. Under the conditions of our experiments, the transition appeared to be due to the more rapid growth of a minor fraction of spontaneously occurring transitioned cells which outgrew the remainder of cells in the population. To obtain the transition, the drug resistance gene must reside on the r-determinants component of the R factor. The transition did not take place when the cells were cultured in medium containing high concentrations of TC. This indicates that the TC resistance genes reside on the resistance transfer factor component of the R factor, which is in agreement with physical studies on R factor deoxyribonucleic acid.     

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.     

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.     

Non-random distribution of transduction termini in transductants from the integrated R plasmid, R100-1

Summary Tra ⁺and tra ^–derivatives of drug resistance plasmid, R100-1, were isolated by phage P1 from an Hfr donor with integrated R100-1 and then analyzed by complementation tests with tra ^–point mutants of Flac. Tra ⁺derivatives of R100-1 carrying tetracycline resistance alone and those carrying all six drug-resistance genes could support transfer of tra ^–point mutants of Flac except Flac traJ, whereas all of tra ^–derivatives of R100-1 failed to complement any one of tra ^–point mutants of Flac. This suggests that these tra ^–derivatives of R100-1 carrying tetracycline resistance gene are deleted for all the transfer genes impaired in the Flac point mutants tested. We assume a hot point, probably a specific base sequence similar to an IS element, at the left of the tetracycline gene (Fig. 1) becomes a transduction terminus in transduction of the integrated R100-1 by phage P1. Complementation analysis of tra ^–derivatives carrying five resistance genes except the tetracycline gene led us to a supposition that a gene(s), probably analogous to traJ of the F plasmid, is located on R100-1 near the tetracycline gene which plays an important regulatory role for self-transfer as well as for the complementation of tra ^–Flac mutants.     

R factor-mediated tetracycline resistance in Escherichia coli K12 dominance of some tetracycline sensitive mutants and relief of dominance by deletion

Summary Strains of Escherichia coli K12 heterozygous for the R100-1 tetracycline resistance region were constructed. They carried the wild-type Tet^r genes in the chromosome and single site Tet^s mutations on plasmids. Some heterozygotes could not express tetracycline resistance fully after induction. The mutant tet allele was thus partially dominant.When heterozygotes carrying the dominant tet mutant were plated on agar containing 50 g/ml tetracycline, mutants which grew normally occurred at a frequency of 1–4×10^-4. Analysis of these dominance relief mutants showed that in 53/56 isolates the dominant tet allele was lost forming either Tra⁺ or Tra^- deletion mutants of the plasmid. The mutation frequency was not affected either by the host chromosomal recA mutation or by the temperature of growth of the culture.     

Molecular Recombination Between R-Factor Deoxyribonucleic Acid Molecules in Escherichia coli Host Cells

THREE PREVIOUSLY STUDIED R FACTORS WERE USED: 222/R4, controlling transmissible resistance to sulfonamide, streptomycin, chloromycetin, and tetracycline (SU(r) SM(r) CM(r) TC(r)); 222/R3, a derivative of 222/R4 (now termed 222/R3W) having lost TC(r); and R15, controlling infectious resistance to SU and SM only. Two types of derivative R factors were isolated from 222/R4 by serial subculture in Salmonella species. One derivative, termed 222/R1, lost resistance to SU, SM, and CM, and the other, termed 222/R3N, lost only TC(r). Each factor was transferred to a standard Escherichia coli K-12 host. Recombinant factors of 222/R4 phenotype were isolated by selection after mixed culture of E. coli (222/R1)(+) and (222/R3N)(+) strains. Density-gradient equilibrium centrifugation of lysates of E. coli R(+) hosts in the presence of ethidium bromide separated R-factor deoxyribonucleic acid (DNA) as a heavy satellite peak which was subjected to electron microscopy or analytical density gradient centrifugation. Each DNA comprised a unimolecular species of circular DNA. The contour of R15 measured 22.3 mum [equivalent to 46 x 10(6) atomic mass units (AMU)], and that of 222/R4 measured 33.6 mum (70 x 10(6) AMU). 222/R3W appeared to be a point mutant or small deletion of 222/R4 with an almost identical size, whereas 222/R3N had lost a DNA segment of about 3 mum, and measured 30.3 mum or 63 x 10(6) AMU. The 222/R1 factors also appeared to have arisen by loss of DNA from 222/R4, 222/R1A being 22.3 mum or 46 x 10(6) AMU, whereas all other 222/R1 factors appeared to be duplicates, measuring 25.6 mum or 53 x 10(6) AMU. The DNA from six recombinant factors of R4 phenotype was indistinguishable in size and configuration from the parental 222/R4. In most cases, the number of R-factor copies (present as covalently closed circular molecules) per copy of the E. coli chromosome was less than 2, ranging from 1.2 to 3.3.     

Drug resistance of Escherichia coli isolated in Nepal.

We collected Escherichia coli strains from 59 Nepalese porters in 1971 and surveyed for their drug resistance. Drug-resistant E. coli strains were isolated from four porters. (TC. CM. SM. SA. APC.)-resistant strains were isolated from two porters and SA- or APC-resistant strains were isolated from each of the others. The R factors were demonstrated from the multiple-resistant E. coli strains.