R-factor Transfer Research Articles

Antimicrobial resistance and R-factor transfer of salmonellae isolated from chicken carcasses in Greek hospitals

A total of 62 salmonellae, belonging to six different serotypes, were isolated from 60 out of 87 (69.0%) chicken carcasses delivered to hospitals of Thesssaloniki, Greece. Salmonella enteritidis, Salmonella anatum and Salmonella bredeney were the most prevalent serovars. Isolates were examined for antibiotic resistance patterns and R-determinants. Resistance to at least one antibiotic was observed in 36 (58.1%) of them and 18 different resistant profiles were recorded. Nitrofurantoin-resistance was the most common (29.0%), followed by spectinomycin (21.0%), ampicillin (19.4%) and ticarcillin (19.4%). Fourteen (38.9%) of the resistant isolates possessed R-factors and resistance to ampicillin, ticarcillin, trimethoprim and kanamycin was easily self-transferable. However, nitrofurantoin- and spectinomycin-resistance although prevailing, was not found transferable even after mobilization. The high incidence of antibiotic resistant salmonellae among chicken carcasses in our hospital setting suggests the need for public health interventions and possible withdrawal of drug selective pressure.

The emergence of antibiotic resistance: Myths and facts in clinical practice

Selection pressure, caused by the use of antibiotics--especially in hospitals--is the main factor responsible for the emergence of antibiotic-resistant bacteria. Resistance can arise endogenously by mutation (one-step, as found for Mycobacterium tuberculosis to rifampicin, or multi-step, as in gonococci to benzylpenicillin), or exogenously by transfer of R-factors. Mechanisms of resistance may involve a decrease in permeability, chemical modification of the antibiotic, or a change in the affinity of the target site. There are many misconceptions concerning the incidence, nature and spread of antibiotic resistance, and some of the most important of these are discussed. The emergence and spread of resistance can be controlled by adhering to antibiotic policies and by preventing or controlling outbreaks of infection. The importance of resistant organisms can be diminished by the development of new antibiotic agents, preferably containing new chemical entities.

Method to Determine Effect of Antibiotics at Residue Levels on R-Factor Transfer

An analytical system was developed which can assess the ability of antibiotic/antimicrobial residues (0.01-1.00 ppm) to affect the conjugal transfer of resistance among the Enterobacteriaceae. The donor strain, Escherichia coli RP-4 (Amr Tcr Nmr Kmr Lac+), and recipient strain, E. coli Sc-8632 (Smr Lac-), were incubated together in a 1:9 donor:recipient ratio for 18 h with gentle shaking (50 rpm) in brain heart infusion broth in the presence of residue levels of antibiotics. The mating cultures were serially diluted and spread-plated onto MacConkey agar containing 25 micrograms streptomycin/mL to select the total recipient population of sensitive E. coli Sc-8632 and transconjugants. After an 18 h incubation at 37 degrees C, the plates were replicated onto MacConkey agar containing 25 micrograms ampicillin/mL to select the ampicillin-resistant transconjugant population. Repeatability was good; the average transfer was 51.8%, with a coefficient of variation of 9.3%. Residue levels of tylosin (0.10 and 1.00 ppm) increased the transfer of the ampicillin marker beyond the 95% confidence limits. Oxytetracycline, bacitracin, streptomycin, penicillin, and virginiamycin did not increase the percent transfer. Oxytetracycline at 0.01 ppm decreased the percent transfer. In general, residue levels of antibiotics (0.01-1.00 ppm) did not affect the conjugal transfer of antibiotic resistance.

Physical map of the conjugal plasmid of Neisseria gonorrhoeae.

The 24.5-megadalton plasmid of Neisseria gonorrhoeae is required for transfer of R-factors and possibly chromosomal markers during conjugal matings between gonococcal strains. We constructed a physical map of one such plasmid, pLE2451, using EcoRI, BglII, and HincII site-specific restriction endonucleases. The patterns of deoxyribonucleic acid digestion obtained with this plasmid were identical to those obtained with three other plasmids of similar size.

Inhibition by Paromomycin of R-Factor Transfer of Tetracycline Resistance between Escherichia coli and Salmonella pullorum

In vitro assays showed that low concentrations (5–10 and 20μg/ml) of the antimicrobial paromomycin sulfate are able to block or diminish significantly the transfer of the tetracycline resistance R-factor between Escherichia coli and Salmonella pullorum. This observation is important because it offers the possibility of preventing the formation of tetracycline-resistant pathogens, a limiting factor of tetracycline use in both human and veterinary medicine.

In vivo inhibition of R-factor transfer by clindamycin and its analogues

In vivo inhibition of R-factor transfer by clindamycin and its analogues

β‐LACTAMASES IN HOSPITAL STRAINS OF GRAM‐NEGATIVE BACTERIA

A collection of 98 strains of enteric Gramnegative bacteria isolated in routine investigations in a hospital laboratory all showed beta-lactamase activity, and 39 of them (40%) produced sufficient enzyme to allow determination of their relative activities against benzylpenicillin, ampicillin and cephaloridine (substrate profiles). The commonest type of beta-lactamase, detected in 33 of the 39 strains, was an "all-purpose" enzyme that showed similar activity against the penicillins and the cephalosporins. All 39 were resistant to multiple antibiotics. They were examined for their ability to transfer the beta-lactamase gene during R-factor transfer, and transferability of the beta-lactamase gene was demonstrated in 13 strains out of 32.

Amplification of sex repressor function of one fi-+ R-factor during anaerobic growth of Escherichia coli.

It has been reported by Mitsuhashi (1965) that transfer of one R-factor was completely inhibited by anaerobic transfer conditions. In contrast, several workers have observed R-factor transfer, although at a reduced rate, in the animal intestines, a largely anaerobic environment. It is shown here that in vitro transfer of the R-factor R1 (F-type pilus, fi-+) in Escherichia coli K-12 is severely depressed, whereas transfer of R64 (I-type pilus, fi-minus) is slightly stimulated by anaerobiosis. Inhibition of R1 fertility is dependent on anaerobic conditions during pregrowth of the donor cells, whereas the oxygen tension during recipient pregrowth, transfer, and plating is of little importance. Anaerobic pregrowth has a less inhibitory effect on the fertility of R1drd19, a mutant of R1 having a defective sex repressor. The fi-+ property of R1 when introduced into F' or Hfr bacteria is amplified during anaerobic growth. These observations strongly indicate that the sex repressor is the mediator of the anaerobic fertility inhibition of the R-factor R1. This hypothesis was supported by studies of the formation of sex pili, the only gene product identified that is controlled by the sex repressor of R1. Using propagation of the F-type pilus-specific phage MS2 as a measure of the degree of sex piliation of a bacterial population, it is shown that in anaerobic cultures sex piliation due to R1 is strongly repressed, whereas piliation due to R1drd19 is repressed to a lesser extent. The possible survival value of the response of R1 towards oxygen tension is discussed.

Antibiotic resistance and R factors in coliform bacilli isolated from hospital and domestic sewage.

SUMMARY One-hundred and one samples from a wide variety of sewers and water-drainage channels in Bristol, including some sewers that separately drained hospital and domestic premises, were examined for antibiotic-resistant coliform bacilli and for R factors. The antibiotic-sensitivity patterns of over 3000 coliform bacilli isolated were obtained and 1000 resistant strains were tested for R-factor transfer. Hospital sewage contained more coliform bacilli with much higher proportions of resistant bacteria, more R factors, and a greater proportion of R factors carrying multiple resistance, than sewage from domestic and other sources. Despite these findings, it was calculated that less than 5% of the R factors in the sewage output of the City of Bristol originate in hospitals. The normal, healthy population appears to be by far the greatest reservoir of R factors in the community.

Chemotherapy and antibiotic-resistance transfer between Enterobacteria in the human gastro-intestinal tract.

SUMMARY The transfer of R factors in the human gastro-intestinal tract and the conditions under which it occurs was studied in healthy persons, who ingested derivatives of Escherichia coli containing chromosomal and R-factor-mediated resistance determinants that had been prepared from strains isolated from their own faeces. The experiments were designed to obtain presumptive evidence of survival and transfer of R factors by studying resistance-marker patterns of organisms isolated from the faeces. To prove conclusively that R-factor transfer had occurred and that the observed marker patterns were not fortuitous, studies of resistance-marker patterns in one of these subjects were supplemented by further detailed investigations including serological and molecular studies, which will be described in the succeeding paper. An incidental finding was a significant positive correlation between the mean number of antibiotic-resistance determinants in resistant organisms and the age of an individual, although these studies were limited to a small and highly selected group of subjects. No evidence of R-factor transfer was obtained in the absence of chemotherapy in four subjects, even though the plasmids concerned could be freely transferred in vitro from the potential donors to a wide range of faecal organisms, including the genetically marked ingested potential recipients. However, treatment of three subjects with a five-day course of a relevant antibiotic after ingestion of R factor bearing organisms led to the appearance in the faeces of large numbers of resistant coliform bacilli with antibiotic-resistance patterns consistent with transfer of the R factor either to an ingested potential recipient organism (one subject) or to endogenous strains of E. coli (two subjects). Chemotherapy greatly prolonged the persistence of ingested R factors in the faecal flora long after the antibiotics were withdrawn and caused the number of R-factor bearing organisms in the faeces to increase. No evidence was obtained in limited studies of fragmentation or loss of R factors from ingested E. coli.

Studies on the nature of plasmids arising from conjugation in the human gastro-intestinal tract.

SUMMARY In the studies described in the preceding paper, circumstantial evidence was found for transfer of antibiotic resistance from an ingested strain of Escherichia coli carrying an R factor, R-1, which determined resistance to ampicillin (A), chloramphenicol (C), kanamycin (K), streptomycin (S) and sulphonamide (Su). Resistant organisms isolated from the faeces of a single individual were examined in detail to prove conclusively that the DNA of the ingested plasmid had transferred to other organisms. The O antigens of the resistant faecal isolates were the same as those of two organisms present before the beginning of the experiment, and these strains were resistant to all the determinants of R-1. The levels of resistance of the isolates to these antibiotics were quantitatively consistent with transfer of the plasmid R-1; and the β-lactamase conferring ampicillin resistance was of the same antigenic type. The predominant resistant organism isolated from the faeces transferred resistance to ACKSSu to laboratory recipients, and plasmid DNA isolated from such strains had the same molecular weight as R-1 and gave 100 per cent. DNA hybridisation with DNA from R-1. Small populations of another strain were also isolated which transferred resistance to ACKSSu and to tetracycline, and this plasmid contained all of the DNA base sequence of R-1 plus additional DNA. The serotype of this strain was the same as that of a strain already resistant to tetracycline before the beginning of the experiment. These results prove that R-factor transfer must have occurred during or after antibiotic therapy and illustrates possible dangers of chemotherapy.

Resistance Factors and Their Ecological Importance to Bacteria and to Man

This chapter explores how man has made the production of antibacterial substances into a multimillion dollar concern; and the selection pressure that this effort exerts on the bacterial population is immense—particularly in certain niches important to man. Transferable antibiotic resistance is merely one example of a general phenomenon, but its study may show the nature of the steps involved in this process and thereby how to mitigate it. Spread certainly occurs: and it frequently goes a great way to undermine the value of new therapeutic agents. The chapter examines the aspects of bacterial genetics and biochemistry that are relevant to the survival and transmission of antibiotic resistance genes between bacterial cells. The chapter discusses the nature and processes associated with the survival and transfer of R-factors and other plasmids in bacteria. The chapter also examines certain examples claiming to show the importance of R-factor-mediated transfer of antibiotic resistance genes during the course of a clinical infection, and try to assess how widespread this process is and how serious in a clinical context. The chapter emphasizes on the aspects of R-factor behavior that have most importance from the ecological point of view and also focuses on the molecular basis of the process.

Requinomycin, an inhibitor of R-factor transfer: isolation, characterization and properties.

A new member of the anthracycline group of antibiotics named requinomycin has been isolated from a strain of Streptomyces filamentosus which had been screened for anti-phage activity. The antibiotic was obtained as yellow crystals, whose molecular formula was assigned to C40H54N2O16. It inhibits the reproduction of phage f 2 in E. coli S-26, the transfer of R-factors between E. coli strains, the growth of Gram-positive bacteria and the growth in vitro of YOSHIDA rat sarcoma cells. Requinomycin resembles aklavin but they are apparently different in antibiotic activity against E. coli B and stability to acid hydrolysis.

Enteropathogenic Bacteria of Nonhuman Primates In vivo and in vitro Transfer of R-Factors from Escherichia coli to Shigella flexneri

Enteropathogenic Bacteria of Nonhuman Primates In vivo and in vitro Transfer of R-Factors from Escherichia coli to Shigella flexneri

Inhibition of R-factor transfer and phage infection by requinomycin.

To be effective in inhibiting R-factor transfer (between Escherichia coli strains) and infection of E. coli with phage f 2, requinomycin must be present at the time of initiation of both events. Delayed addition of requinomycin showed no or slight inhibition. No direct effect of requinomycin on phage f2 or E. coli strains employed was observed. The intact cell surface of E. coli must be impermeable to requinomycin as shown by the fact that DNA synthesis by toluene-treated E. coli cells (permeability modified) was markedly inhibited by requinomycin, in contrast to its ineffectiveness on intact E. coli cells. Therefore, requinomycin probably acts without penetrating the cells. Probable mechanism of action is discussed.

Inhibition of R-factor transfer by levallorphan

The transfer of an R-factor from Proteus Rettgeri to Escherichia coli W 677 was strongly inhibited by levallorphan at concentrations which do not affect growth of either the donor or the recipient cells. The effect is not due to the transfer of the episom itself but to inhibition of mating-pair formation.

R factors in strains of Shigella dysenteriae type 1 isolated in the western hemisphere during 1969-1970.

Eighteen strains of Shigella dysenteriae type 1 isolated at various times and localities during the epidemic of dysentery in Central America and Mexico during 1969-1970 were examined for characterization of the multiple antibiotic resistance of these strains and determination of whether transferable resistance factors were present. Seventeen strains were resistant to sulfonamides, streptomycin, tetracycline, and chloramphenicol, and one was resistant to sulfonamides, streptomycin, and chloramphenicol. All strains transferred the complete pattern of resistance to Escherichia coli. Nearly all R+ recipients examined, regardless of the antibiotic with which they were selected, exhibited the complete resistance pattern of the donor. Frequency of R-factor transfer was similar in the four strains in which it was determined. The levels of resistance conferred by these R factors were similar in both S. dysenteriae and E. coli for all drugs except streptomycin (minimal inhibitory concentration, 125-250 fxg/ml in S. dysenteriae and 8-16 \igZm in E. coli). The findings of this study suggest that the multi-resistant strains isolated in this epidemic harbor the same R factor rather than a heterogenous group of drug-resistance factors of diverse origins.

Increased resistance to several antibiotics by one mutation in an R-factor, R 1a.

SUMMARY: An Hfr strain G11 and an F- strain D1 of Escherichia coli K 12 both containing the R-factor R 1a were treated with ethyl methane sulphonate (EMS). Mutation of the bacteria to high ampicillin resistance occurred at a frequency of 10-3 to 10-4 and it was coupled with a simultaneous increase in resistance to chloramphenicol, streptomycin and sulphanilamide. Resistance to all the antibiotics was infective. Increased ampicillin resistance was due to increased penicillinase activity of the bacteria. In a strain containing a mutant R-factor (R1 B1) this was due to an increased quantity of an enzyme that seemed to be identical to that of a strain containing the unmutated R 1a. Metabolism of chloramphenicol in strains containing R1 B1 was greater than in those with R 1a. Mating experiments with an Hfr strain carrying R 1a or R1 B1 revealed that the two R-factors reduced fertility to about the same extent. Pair formation was two to three times greater in the presence of R1 B1 than in the presence of R 1a, and R-factor transfer was increased more than chromosome transfer. It is likely that the R 1a genome is read two to three times more efficiently in strains containing the mutated R1 B1, perhaps due to an increased number of copies of the R-factor genome per bacterium.

R-Factor Transfer in Selenite and Tetrathionate Broths

After overnight incubation of R(+)Escherichia coli with R(-)Salmonella typhimurium in selenite and tetrathionate with Brilliant Green (TBG) broths, R-factor transfer was demonstrated in 10 of 12 experiments. R-factor transfer in these enrichment broths occurred at a markedly reduced frequency in comparison to that in Trypticase soy broth, apparently due to an adverse effect either on the viability of the donor E. coli or the conjugation process itself. Transfer of R factors in commonly used enrichment broths may give rise to falsely resistant antibiotic patterns in Salmonella. However, the frequency of R-factor transfer is so low that it is unlikely to affect significantly the interpretation of R-factor studies.

Effect of rifampicin on stability and transfer of R-factors.

Rifampicin LEPETIT, an antibiotic known to interfere with the activity of DNA-dependent bacterial RNA polymerase, prevented the conjugational transfer of R-factors in Salmonella typhimurium and Escherichia coli, and showed appreciable accumulation of R— variants in R+ cultures. The drug was active in concentrations from 0.25 to 5.0 μg/ml while there was no influence on the viable count of the R+ cultures. Although the concentrations of rifampicin found to be active in R+ strains of enterobacteria are about 10 times higher than those found to be active in staphylococci by JOHNSTON and RICHMOND (1970), the sub-bacteriostatic concentrations of the drug were effective enough to eliminate R-factors and to prevent their transfer from the donor cells studied.