Expression Of Antibiotic Resistance Genes Research Articles

Emergence of Plasmid-Mediated Quinolone Resistance in Escherichia coli in Europe

Although quinolone resistance results mostly from chromosomal mutations, it may also be mediated by a plasmid-encoded qnr gene in members of the family Enterobacteriaceae. Thus, 297 nalidixic-acid resistant strains of 2,700 Escherichia coli strains that had been isolated at the Bicetre Hospital (Le Kremlin-Bicetre, France) in 2003 were screened for qnr by PCR. A single E. coli isolate that carried a ca. 180-kb conjugative plasmid encoding a qnr determinant was identified. It conferred low-level resistance to quinolones and was associated with a chromosomal mutation in subunit A of the topoisomerase II gene. The qnr gene was located on a sul1-type class 1 integron just downstream of a conserved region (CR) element (CR1) comprising the Orf513 recombinase. Promoter sequences for qnr expression overlapped the extremity of CR1, indicating the role of CR1 in the expression of antibiotic resistance genes. This integron was different from other qnr-positive sul1-type integrons identified in American and Chinese enterobacterial isolates. In addition, plasmid pQR1 carried another class 1 integron that was identical to In53 from E. coli. The latter integron possessed a series of gene cassettes, including those coding for the extended-spectrum beta-lactamase VEB-1, the rifampin ADP ribosyltransferase ARR-2, and several aminoglycoside resistance markers. This is the first report of plasmid-mediated quinolone resistance in Europe associated with an unknown level of plasmid-mediated multidrug resistance in Enterobacteriaceae.

Persistence of plasmid RP4 in Pseudomonas putida and loss of its expression of antibiotic resistance in a groundwater microcosm

We examined the stability of plasmid RP4 and its expression of antibiotic-resistance genes in suspended and sorbed Pseudomonas putida in aquifer microcosms. Test tubes containing different proportions of sterilized aquifer soil and groundwater were inoculated with bacteria and incubated for up to 26 d. Serial dilutions were made to agar plates with or without antibiotics, to quantify the functional stability of the plasmid. The structural integrity of RP4 was examined by plasmid extraction, digestion with restriction enzymes, and agarose gel electrophoresis. The plasmid-borne resistance gene expression disappeared in 80–90% of the cells during day 1 of incubation in aquifer soil and then remained at that frequency throughout the experiment. The RP4 plasmid was present in cells without antibiotic-resistance gene expression, indicating that the observed loss of plasmid-encoded activity was most likely due to a reduction in expression of the resistance genes. The increased growth rate in groundwater amended with glucose and phosphate had no significant influence on plasmid loss or antibiotic-resistance expression, suggesting that plasmid loss and antibiotic-resistance expression were independent of the growth rate. Most of the reduction of resistance gene expression was associated with the presence of soil particles, and 70% of the resistance expression was retained in bacteria incubated for 1 d in groundwater alone. Bacteria sorbed to the soil particles had a lower frequency of expression of resistance genes than suspended bacteria, but the difference was not caused by sorbed inorganic or organic chemicals. Resistance gene expression was partly recovered in suspended bacteria after in vitro exposure to the antibiotics and after first isolating on agar without antibiotics and then replica plating to agar containing the antibiotics.

Persistence of plasmid RP4 in Pseudomonas putida and loss of its expression of antibiotic resistance in a groundwater microcosm

We examined the stability of plasmid RP4 and its expression of antibiotic-resistance genes in suspended and sorbed Pseudomonas putida in aquifer microcosms. Test tubes containing different proportions of sterilized aquifer soil and groundwater were inoculated with bacteria and incubated for up to 26 d. Serial dilutions were made to agar plates with or without antibiotics, to quantify the functional stability of the plasmid. The structural integrity of RP4 was examined by plasmid extraction, digestion with restriction enzymes, and agarose gel electrophoresis. The plasmid-borne resistance gene expression disappeared in 80–90% of the cells during day 1 of incubation in aquifer soil and then remained at that frequency throughout the experiment. The RP4 plasmid was present in cells without antibiotic-resistance gene expression, indicating that the observed loss of plasmid-encoded activity was most likely due to a reduction in expression of the resistance genes. The increased growth rate in groundwater amended with glucose and phosphate had no significant influence on plasmid loss or antibiotic-resistance expression, suggesting that plasmid loss and antibiotic-resistance expression were independent of the growth rate. Most of the reduction of resistance gene expression was associated with the presence of soil particles, and 70% of the resistance expression was retained in bacteria incubated for 1 d in groundwater alone. Bacteria sorbed to the soil particles had a lower frequency of expression of resistance genes than suspended bacteria, but the difference was not caused by sorbed inorganic or organic chemicals. Resistance gene expression was partly recovered in suspended bacteria after in vitro exposure to the antibiotics and after first isolating on agar without antibiotics and then replica plating to agar containing the antibiotics.

Overexpression of the multidrug efflux pump SmeDEF impairs Stenotrophomonas maltophilia physiology.

The use of antibiotics for the treatment of infectious diseases has led to important changes in the structure of pathogenic bacterial populations. However, these changes could be buffered if the expression of antibiotic resistance genes were to lead to the counter-selection of antibiotic-resistant strains in antibiotic-free environments. To test the effect of antibiotic resistance on bacterial fitness, we analysed the effect of the overproduction of the multidrug efflux pump SmeDEF on the physiology of Stenotrophomonas maltophilia. SmeDEF confers resistance to antibiotics belonging to different structural families, and its overexpression is associated with an antibiotic resistance phenotype in clinical isolates of S. maltophilia. Two S. maltophilia isogenic strains were analysed: the wild-type strain D457 and strain D457R, which is a SmeDEF overproducer. In co-culture experiments, under non-selective pressure the wild-type strain displaced the mutant strain D457R. Metabolic profiling showed that SmeDEF overproduction leads to several changes in S. maltophilia metabolism. Using a Dictyostelium discoideum model of bacterial virulence, we found overexpression of SmeDEF to be associated with a reduction in S. maltophilia virulence. Together, these data indicate that overexpression of the multidrug efflux pump SmeDEF causes a metabolic burden for S. maltophilia.

Expression of antibiotic resistance genes in the integrated cassettes of integrons

Plasmids containing cloned integron fragments which differ only with respect to either the sequence of the promoter(s) or the number and order of inserted cassettes were used to examine the expression of resistance genes encoded in integron-associated gene cassettes. All transcripts detected commenced at the common promoter P(ant), and alterations in the sequence of P(ant) affected the level of resistance expressed by cassette genes. When both P(ant) and the secondary promoter P2 were present, transcription from both promoters was detected. When more than one cassette was present, the position of the cassette in the array influenced the level of antibiotic resistance expressed by the cassette gene. In all cases, the resistance level was highest when the gene was in the first cassette, i.e., closest to P(ant), and was reduced to different extents by the presence of individual upstream cassettes. In Northern (RNA) blots, multiple discrete transcripts originating at P(ant) were detected, and only the longer transcripts contained the distal genes. Together, these data suggest that premature transcription termination occurs within the cassettes. The most abundant transcripts appeared to contain one or more complete cassettes, and is possible that the 59-base elements found at the end of the cassettes (3' to the coding region) not only function as recombination sites but may also function as transcription terminators.

Stable expression of antibiotic resistance genes using a promoter fragment of the U1 snRNA gene

As U1 snRNA is produced in all mammalian cell types, antibiotic resistance genes driven by this promoter would be ideally suited as genetic selection markers. However, although the U1 snRNA gene is transcribed by RNA polymerase II, its native product is not a messenger RNA, but a splicing cofactor. To test whether this promoter could nevertheless produce a functional mRNA, sensitive reporter genes expressing resistance to the antibiotics hygromycin-B and bleomycin were constructed with either the U1 snRNA promoter or the SV40 early promoter. Resistant cell lines could only be obtained with constructs equipped with a functional polyadenylation signal. With the U1 snRNA promoter about three times fewer colonies were obtained than with the SV40 early promoter. Another potential advantage of the U1 snRNA promoter is that, in contrast to the promoters commonly used to express genetic selection markers, the enhancer-like element contained in the U1 snRNA promoter had only a minimal stimulative effect, only detectable with the most sensitive methods, on an adjacent mRNA-producing gene. The U1 snRNA promoter was also capable of expressing bleomycin resistance in the context of a self-inactivating retrovirus vector, whereby it was discovered that the mouse 3T3 cells used in this experiment were 10 times more sensitive to bleomycin than human or hamster cell lines.