A differential drug screen for compounds that select against antibiotic resistance

Antibiotics increase the frequency of resistant bacteria by providing them a competitive advantage over sensitive strains. Here, we develop a versatile assay for differential chemical inhibition of competing microbial strains, and use it to identify compounds that preferentially inhibit tetracycline-resistant relative to sensitive bacteria, thus "inverting" selection for resistance. Our assay distinguishes compounds selecting directly against specific resistance mechanisms and compounds whose selection against resistance is based on their physiological interaction with tetracycline and is more general with respect to resistance mechanism. A pilot screen indicates that both types of selection-inverting compounds are secreted by soil microbes, suggesting that nature has evolved a repertoire of chemicals that counteracts antibiotic resistance. Finally, we show that our assay can more generally permit simple, direct screening for drugs based on their differential activity against different strains or targets.     

The expression of Streptomyces and Escherichia coli drug-resistance determinants cloned into the Streptomyces phage phi C31

Lysogens obtained by infecting Streptomyces albus G with a phi C31-pBR322 chimaeric prophage or its delta W12 deletion derivative had increased tetracycline resistance. The ability of the delta W12 derivative to transduce tetracycline resistance was inactivated by inserting a viomycin resistance determinant (vph) into the BamHI site of the pBR322 tet gene, and restored by excising the vph gene. Another deletion mutant (delta W17) of the chimaera, carrying an intact tet gene, was normally unable to transduce tetracycline resistance. This inability was correlated with the finding, by Southern hybridisation analysis, that the att site required for insertion of phi C31 prophage into the host chromosome was located within the delta W17 deletion. Use of phi C31 lysogenic recipient permitted the integration of the att-deleted phage, presumably by homologous recombination, giving tetracycline-resistant double lysogens. This technique was extended to S. coelicolor A3(2) in the detection of derivatives of the att-deleted phage into which a thiostrepton-resistance determinant (tsr) had been inserted in vitro. Phage released from double lysogens were mainly recombinants. One such recombinant is a PstI vector for DNA cloning, able to accommodate up to 6 kb of introduced DNA.     

Uptake of 3H-Tetracycline by Resistant and Sensitive Escherichia coli

The uptakes of (3)H-tetracycline by 12 tetracycline-sensitive and 24 tetracycline-resistant Escherichia coli hospital cultures were found to be 270 and 75 nmoles of tetracycline per milliliter of cell water per 20 min, respectively. This confirms reports by other investigators who, by using only one or two cultures, suggested a relationship between tetracycline uptake and tetracycline resistance. However, minimum inhibitory concentrations of tetracycline for the cultures bore no relation to the tetracycline uptake values, suggesting that loss of tetracycline uptake may not be the primary cause of resistance. In addition there were three resistant cultures with uptake values greater than 140 and two sensitive cultures with uptakes lower than 180, raising the question of how these tetracycline-resistant cultures could grow with tetracycline at concentrations nearly as high as those found to inhibit growth of sensitive organisms. Of the tetracycline-resistant cultures, 15 were able to transfer tetracycline resistance to a recipient organism and 9 were not. Two of the cultures transferred TC-resistance to a recipient with no modification-restriction system (E. coli C) but did not transfer resistance to a recipient with a known modification-restriction system (E. coli K-12).     

Clustering of mutations affecting alginic acid biosynthesis in mucoid Pseudomonas aeruginosa.

A 10-kilobase DNA fragment previously shown to contain the phosphomannose isomerase gene (pmi) of Pseudomonas aeruginosa was used to construct a pBR325-based hybrid that can be propagated in P. aeruginosa only by the formation of a chromosomal-plasmid cointegrate. This plasmid, designated pAD4008, was inserted into the P. aeruginosa chromosome by recombination at a site of homology between the cloned P. aeruginosa DNA and the chromosome. Mobilization of pAD4008 into P. aeruginosa PAO and 8830 and selection for the stable acquisition of tetracycline resistance resulted in specific and predictable changes in the pattern of endonuclease restriction sites in the phosphomannose isomerase gene region of the chromosomes. Chromosomal DNA from the tetracycline-resistant transformants was used to clone the drug resistance determinant with Bg/II or XbaI, thereby allowing the "walking" of the P. aeruginosa chromosome in the vicinity of the pmi gene. Analysis of overlapping tetracycline-resistant clones indicated the presence of sequences homologous to the DNA insert of plasmid pAD2, a recombinant clone of P. aeruginosa origin previously shown to complement several alginate-negative mutants. Restriction mapping, subcloning, and complementation analysis of a 30-kilobase DNA region demonstrated the tight clustering of several genetic loci involved in alginate biosynthesis. Furthermore, the tetracycline resistance determinant in PAO strain transformed by pAD4008 was mapped on the chromosome by plasmid FP2-mediated conjugation and was found to be located near 45 min.     

Genetic recombination of bacterial plasmid DNA. Physical and genetic analysis of the products of plasmid recombination in Escherichia coli

Derivatives of plasmid pBR322 DNA containing tet mutations were constructed by inserting XhoI linkers at various sites in the tetracycline resistance gene. Monomer plasmids containing either the tet-10 allele located at nucleotide position 23 or the tet-14 allele located at nucleotide position 1267 were used to construct a circular dimer containing one copy of each allele and a circular trimer containing one copy of the tet-10 allele and two copies of the tet-14 allele. Genetic recombination of these plasmid DNAs to produce a functional tetracycline resistance gene could be detected as the production of tetracycline-resistant progeny during the growth of transformants or using a restriction mapping assay which detected the rearrangement of the mutant alleles. The structure of individual tetracycline-resistant recombination products was determined by restriction mapping. This analysis suggested that as many as 70% of the plasmid recombination events in Escherichia coli AB1157 could have involved gene conversion events. The formation of these recombination products was most easily predicted by a model involving figure 8 recombination intermediates and the formation of symmetric regions of heteroduplex. Recombination in JC10287 delta(srlR-recA)304 occurred at 5% of the wild-type frequency and appeared to occur by a similar mechanism. Recombination in JC9604 recA56 recB21 recC22 sbcA23 occurred at 20 times the wild-type frequency and appeared to involve multiple independent recombination events.     

A quantitative real-time PCR assay for the detection of tetR of Tn10 in Escherichia coli using SYBR Green and the Opticon

Bacteria of implant infections are extremely resistant to antibiotics. One reason for this antibiotic resistance are transposons; the well-known transposon Tn10, for example, mediates tetracycline resistance to Escherichia coli. Two genes of Tn10, tetA and tetR, are essential for the mechanism of resistance. These genes encode a drug-specific efflux protein and a tetracycline repressor protein, respectively. Tn10 is also widely used in molecular biology. For example, tTA, a recombinant derivate of tetR, has been utilised for a highly efficient gene regulation system in mammalian cells. We have examined E. coli isolates from implant infections for tetracycline resistance and for the presence of tetR. A real-time PCR assay was developed for detection of tetR with SybrGreen using the Opticon PCR machine of MJ Research. This method offers a quick, sensitive, efficient, and reliable approach to the detection and quantification of genes. Clinical isolates of E. coli were examined successfully for tetracycline resistance and for the presence of tetR. The real-time PCR is effective using a variety of templates including isolated E. coli DNA, pure colonies, or liquid culture sources. Using quantified standard DNA, this assay can accurately detect as few as 15 copies. Moreover, this assay has the ability to quantify the number of tetR genes in the presence of contaminating mammalian DNA. In conclusion, the tetR real-time PCR offers new methods for detection and quantification of tetracycline-resistant bacteria and tTA in transfected cell-lines or transgenic animals.     

Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage

The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tc(r)) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the tet genes, conferring tetracycline resistance, that they carry. With the (GTG)(5)-PCR DNA fingerprinting technique, the Tc(r) lactic acid bacterial isolates were identified as Lactobacillus plantarum, L. sakei subsp. carnosus, L. sakei subsp. sakei, L. curvatus, and L. alimentarius and typed to the intraspecies level. For a selection of 24 Tc(r) lactic acid bacterial isolates displaying unique (GTG)(5)-PCR fingerprints, tet genes were determined by means of PCR, and only tet(M) was detected. Restriction enzyme analysis with AccI and ScaI revealed two different tet(M) allele types. This grouping was confirmed by partial sequencing of the tet(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with tet(M) genes previously reported in Staphylococcus aureus MRSA 101 and in Neisseria meningitidis, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained tet(M)-carrying plasmids. In addition to the tet(M) gene, one isolate also contained an erm(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the tet(M) genes were not located on transposons of the Tn916/Tn1545 family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse Lactobacillus strains from different types of fermented meat products can be a host for plasmid-borne tet genes.     

Molecular analysis of tetracycline-resistant gonococci: rapid detection of resistant genotypes using a real-time PCR assay

The aim of this study was to examine tetracycline-resistant gonococci and to set up a real-time PCR method to identify, in the same assay, both the chromosomally and the plasmid-mediated tetracycline-resistant genotypes. A retrospective analysis for tetracycline susceptibility was performed by the E -test and agar dilution methods on 289 gonococci isolated in Italy from 2003 to 2005. Molecular mechanisms of resistance were investigated by both sequence analyses of the three main genes associated with chromosomally mediated resistance ( mtrR , penB and rpsJ genes) and by the identification of plasmids carrying the tet M determinant associated with plasmid-mediated resistance, by PCR (American- or Dutch-type plasmids). The genetic relatedness of nonsusceptible strains was evaluated by pulsed field gel electrophoresis (PFGE). The results showed the presence of 22.5% tetracycline-resistant and 49.5% tetracycline-intermediate gonococci. Coexistence of chromosomally and plasmid-mediated resistance to tetracycline was observed in the majority of resistant isolates. No clonal structure was highlighted by analysis of PFGE pattern profiles. Real-time PCR assay was able to identify all the tetracycline nonsusceptible gonococci correctly for the presence of both chromosomally and/or plasmid-mediated genotypes.     

Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance.

Tetracyclines were discovered in the 1940s and exhibited activity against a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. They are inexpensive antibiotics, which have been used extensively in the prophlylaxis and therapy of human and animal infections and also at subtherapeutic levels in animal feed as growth promoters. The first tetracycline-resistant bacterium, Shigella dysenteriae, was isolated in 1953. Tetracycline resistance now occurs in an increasing number of pathogenic, opportunistic, and commensal bacteria. The presence of tetracycline-resistant pathogens limits the use of these agents in treatment of disease. Tetracycline resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines or for a protein that protects bacterial ribosomes from the action of tetracyclines. Many of these genes are associated with mobile plasmids or transposons and can be distinguished from each other using molecular methods including DNA-DNA hybridization with oligonucleotide probes and DNA sequencing. A limited number of bacteria acquire resistance by mutations, which alter the permeability of the outer membrane porins and/or lipopolysaccharides in the outer membrane, change the regulation of innate efflux systems, or alter the 16S rRNA. New tetracycline derivatives are being examined, although their role in treatment is not clear. Changing the use of tetracyclines in human and animal health as well as in food production is needed if we are to continue to use this class of broad-spectrum antimicrobials through the present century.     

A Phage-Mediated Transfer of Chromosomally Integrated Tetracycline Resistance Plasmid in Staphylococcus aureus

The ability of a Staphylococcus aureus isolate WBG7416 to transfer its resistance determinants was studied in conjugation and mixed-culture transfer experiments. It carried plasmid-borne resistance to kanamycin, trimethoprim, chloramphenicol, cadmium, propamidine isethionate, and chromosomal resistance to methicillin, gentamicin, streptomycin, erythromycin, clindamycin, tetracycline, and minocycline. It transferred tetracycline resistance in mixed-culture transfer but not in conjugation experiments. DNA-DNA hybridization of genomic DNA from the tetracycline-resistant transferrants against a labeled tetracycline resistance plasmid, pWBG3, probe revealed the presence of an integrated plasmid in their chromosomes. In contrast, no homology to the probe was detected in the chromosome of a tetracycline-resistant mutant of the recipient strain. The results established a role for a bacteriophage in the transfer of chromosomal tetracycline resistance in WBG7416 besides demonstrating the presence of an integrated tetracycline resistance plasmid in the transferrants. It also offered an insight into the nature of the integrated plasmid.     

The isolation of tetracycline-resistant strains of strictly anaerobic bacteria from the rumen

Tetracycline resistant (Tc^R) strains of three of the major species of strictly anaerobic rumen bacteria Megasphaera elsdenii, Selenomonas ruminantium and Butyrivibrio fibrisolvens , were recovered with an isolation medium containing 20 μg/ml tetracycline. Only two of 14 strains of these species from other sources, isolated without antibiotic selection, showed tetracycline resistance. Evidence was found for the presence of plasmids in two tetracycline-resistant strains of M. elsdenii , and in some strains of S. ruminantium.     

Evidence that a novel tetracycline resistance gene found on two Bacteroides transposons encodes an NADP-requiring oxidoreductase.

Two transposons, Tn4351 and Tn4400, which were originally isolated from the obligate anaerobe Bacteroides fragilis, carry a tetracycline resistance (Tcr) gene that confers resistance only on aerobically grown Escherichia coli. This aerobic Tcr gene, designated tetX, has been shown previously to act by chemically modifying tetracycline in a reaction that appears to require oxygen. We have now obtained the DNA sequence of tetX and 0.6 kb of its upstream region from Tn4400. Analysis of the DNA sequence of tetX revealed that this gene encoded a 43.7-kDa protein. The deduced amino acid sequence of the amino terminus of the protein had homology with a number of enzymes, all of which had in common a requirement for NAD(P). In an earlier study, we had observed that disrupted cells, unlike intact cells, could not carry out the alteration of tetracycline. We have now shown that if NADPH (1 mM) is added to the disrupted cell preparation, alteration of tetracycline occurs. Thus, TetX appears to be an NADP-requiring oxidoreductase. Tn4400 conferred a fivefold-lower level of tetracycline resistance than Tn4351. This finding appears to be due to a lower level of expression of the tetX on Tn4400, because the activity of a tetX-lacZ fusion from Tn4400 was 10-fold lower than that of the same fusion from Tn4351. A comparison of the sequence of the tetX region on Tn4351 with that on Tn4400 showed that the only difference between the upstream regions of the two transposons was a 4-base change 350 bp upstream of the start of the tetX coding region. The 4-base change difference creates a good consensus -35 region on Tn4351 that is not present on Tn4400 and could be creating an extra promoter.     

A broad-host-range plasmid for isolating mobile genetic elements in gram-negative bacteria

Plasmid pGBG1 was constructed to isolate mobile genetic elements in a wide variety of gram-negative bacteria. The mutation target, carried on a broad-host-range vector, allows positive selection for tetracycline resistance. In tests using several gram-negative bacteria we could detect transposition events of either insertion sequences or transposons. A new insertion sequence (IS) element was identified in Ralstonia eutropha.     

Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food

The genetic determinants responsible for the resistances against the antibiotics tetracycline [tet(M), tet(O), tet(S), tet(K) and tet(L)], erythromycin (ermA,B,C; mefA,E; msrA/B; and ereA,B) and chloramphenicol (cat) of 38 antibiotic-resistant Enterococcus faecium and Enterococcus faecalis strains from food were characterised. In addition, the transferability of resistance genes was also assessed using filter mating assays. The tet(L) determinant was the most commonly detected among tetracycline-resistant enterococci (94% of the strains), followed by the tet(M) gene, which occurred in 63.0% of the strains. Tet(K) occurred in 56.0% of the resistant strains, while genes for tet(O) and tet(S) could not be detected. The integrase gene of the Tn916-1545 family of transposons was present in 81.3% of the tetracycline resistant strains, indicating that resistance genes might be transferable by transposons. All chloramphenicol-resistant strains carried a cat gene. 81.8% of the erythromycin-resistant strains carried the ermB gene. Two (9.5%) of the 21 erythromycin-resistant strains, which did not contain ermA,B,C, ereA,B and mphA genes harboured the msrC gene encoding an erythromycin efflux pump, which was confirmed by sequencing the PCR amplicon. In addition, all E. faecium strains contained the msrC gene, but none of the E. faecalis strains. Transfer of the genetic determinants for antibiotic resistance could only be demonstrated in one filter mating experiment, where both the tet(M) and tet(L) genes were transferred from E. faecalis FAIR-E 315 to the E. faecalis OG1X recipient strain. Our results show the presence of various types of resistance genes as well as transposon integrase genes associated with transferable resistances in enterococci, indicating a potential for gene transfer in the food environment.     

A novel type of transposon generated by insertion element Is102 present in a psc101 derivative

We describe a novel type of transposon in the tetracycline resistance plasmid pYM103, a derivative of pSC101 carrying a single copy of an insertion element IS102. The new transposons we found were identified as DNA segments, approximately 6 kb (Tn1021) and 10 kb (Tn1022) in length, able to mediate the cointegration of pYM1O3 with plasmid Col E1. The resulting cointegrate contains either of these pYM1O3 segments duplicated in a direct orientation at the junctions of the parent plasmids. A direct duplication of a 9 bp sequence at the target site in Col E1 is found at the junctions for cointegration. Both transposons have IS1O2 at one end and also contain different lengths of the pYM103 DNA adjacent to IS102, including the tetracycline resistance gene. Each transposon contains terminal inverted repeats of a short nucleotide sequence. These results and the fact that IS102 can itself mediate plasmid cointegration, giving rise to a duplication of a 9 bp target sequence, indicate that IS102 is responsible for generation of Tn1021 and Tn1022. They are quite different from the common IS-associated transposons, which are always flanked by two copies of an IS element, and may be similar to transposons such as those of the Tn3 family and phage Mu.     

Acquired tetracycline and/or macrolide-lincosamides-streptogramin resistance in anaerobes

In general bacterial antibiotic resistance is acquired on mobile elements such as plasmids, transposons and/or conjugative transposons. This is also true for many antibiotic resistant anaerobic species described in the literature. Of the 23 different tetracycline resistant efflux genes identified, tet(B), tet(K), tet(L), and tetA(P) have been found in anaerobic species and six of the ten tetracycline resistant genes coding for ribosomal protection proteins, tet(M), tet(O), tetB(P), tet(Q), tet(W), and tet(32), have been identified in anaerobes. There are now three enzymes which inactivate tetracycline, of which the tet(X) has been identified in Bacteroides though is not functional under anaerobic growth conditions. A similar situation exists with the genes conferring macrolide-lincosamide-streptogramin (MLS) resistance. Of the 26 rRNA methylase MLS resistant genes characterized, five genes; erm(B), erm(C), erm(F), erm(G), and erm(Q), have been identified in anaerobes. In contrast, no genes coding for MLS resistant efflux proteins or inactivating enzymes have been described in anaerobic species. This mini-review will summarize what is known about tetracycline and MLS resistance in genera with anaerobic species and the mobile elements associated with acquired tetracycline and/or MLS resistance genes.     

Evidence for natural horizontal transfer of tetO gene between Campylobacter jejuni strains in chickens

AIMS: The transfer of tetO gene conferring resistance to tetracycline was studied between Campylobacter jejuni strains, in the digestive tract of chickens. METHODS AND RESULTS: In vitro conjugation experiments were first performed in order to select donor/recipient couples for further in vivo assay. Then, chickens were inoculated with a donor/recipient couple of C. jejuni strains displaying spontaneous in vitro tetracycline resistance gene transfer. The donor was a tetracycline-resistant ampicillin-susceptible strain, and the recipient was a tetracycline-susceptible ampicillin-resistant strain. Chicken droppings were streaked on antimicrobial selective media and bi-resistant Campylobacter isolates were further characterized according to their donor or recipient flaA gene RFLP profile. The acquisition of tetracycline-resistance gene by the recipient C. jejuni strain from the donor C. jejuni strain was confirmed by tetO PCR. CONCLUSIONS: The study showed that transfer of tetO gene occurs rapidly and without antimicrobial selection pressure between C. jejuni strains in the digestive tract of chickens. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid and spontaneous transfer of tetO gene may explain the high prevalence of tetracycline resistance in chicken Campylobacter strains.     

Influence of tetracycline exposure on tetracycline resistance and the carriage of tetracycline resistance genes within commensal Escherichia coli populations

AIMS: To assess the influence of incremental tetracycline exposure on the genetic basis of tetracycline resistance within faecal Escherichia coli. METHODS AND RESULTS: Through the adoption of a novel combination of multiple breakpoint selection, phenotypic characterization and the application of a polymerase chain reaction based gene identification system it proved possible to monitor the influence of antibiotic exposure on resistance gene possession. Using tetracycline as a case study a clear hierarchy was revealed between tet genes, strongly influenced by host antimicrobial exposure history. CONCLUSIONS: The antimicrobial exposure regime under which an animal is produced affects both the identity and magnitude of resistance gene possession of a selected bacterial population within its enteric microflora. Among the ramifications associated with such resistance gene selection is the degree of resistance conferred and the carriage of linked resistance determinants. This selection is applied by exposure to antibiotic concentrations well below recognized minimum inhibitory tetracycline concentration breakpoints widely adopted to characterize bacterial 'susceptibility'. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the ability of minimal antibiotic exposure to select for the continued persistence of resistance genes within the enteric microflora. It is clearly demonstrated that different antimicrobial regimes select for different resistance genes, the implications of which are discussed.     

Molecular analysis of tetracycline resistance in Salmonella enterica subsp. enterica serovars Typhimurium, enteritidis, Dublin, Choleraesuis, Hadar and Saintpaul: construction and application of specific gene probes

A total of 65 epidemiologically unrelated tetracycline-resistant isolates of the six Salmonella enterica subsp. enterica (Salm.) serovars Dublin, Choleraesuis, Typhimurium, Enteritidis, Hadar and Saintpaul were investigated for the presence of tetracycline resistance genes. For this, specific gene probes of the tetracycline resistance genes (tet) of the hybridization classes A, B, C, D, E and G were constructed by cloning PCR-amplified internal segments of the respective tet structural genes. These gene probes were sequenced and used in hybridization experiments with plasmid DNA or endonuclease digested whole cell DNA as targets. Only tet(A) genes were detected on plasmids in all Salm. Dublin isolates as well as in single isolates of Salm. Choleraesuis and Salm. Typhimurium. Genes of the hybridization classes B, C, D and G, but also in some cases those of class A, were located in the chromosomal DNA of the corresponding Salmonella isolates. Restriction fragment length polymorphisms (RFLPs) of tet gene carrying fragments were detected in chromosomally tetracycline-resistant isolates. These RFLPs might represent valuable additional tools for the identification and characterization of tetracycline-resistant Salmonella isolates.