Genetic analysis of drug resistance in Neisseria gonorrhoeae: production of increased resistance by the combination of two antibiotic resistance loci.

The studies reported here demonstrate that increased resistance of Neisseria gonorrhoeae to penicillin, tetracycline, and chloramphenicol results from the combined effect of two resistance loci. As shown by experiments with deoxyribonucleic acid from transformants carrying only a single resistance locus, transformants with an incresed level of resistance to penicillin result from the combination of a penicillin-specific locus, pen, and a multiple resistance locus, mtr. Similarly, transformants with an increased level of resistance to tetracycline result from the combination of mtr and a tetracycline-specific locus, tet. Transformants with an increased level of resistance to chloramphenicol result from the combination of mtr and a chloramphenicol-specific locus, cml. Deoxyribonucleic acid dilution experiments established that only a single dose of each of the two required resistance loci is necessary to give higher-level resistance. Higher-level-resistant transformants were not obtained when a double dose of one resistance locus or a combination of loci pairs other than mtr and pen, mtr and tet, or mtr and cml was introduced into a recipient. Combinations of the mtr and tet genes resulted in increased resistance to semisynthetic tetracyclines. The presence of the mtr and pen genes resulted in increased resistance to penicillinase-stable penicillins.     

An N-terminal domain of the tetracycline resistance protein increases susceptibility to aminoglycosides and complements potassium uptake defects in Escherichia coli

Expression of extrachromosomal tet genes increased the susceptibility of gram-negative bacteria to specific aminoglycoside antibiotics. The magnitude of the increase in susceptibility was dependent on the amount and the class of the tet gene product (designated Tet) and the bacterial species in which the tet gene was expressed. Truncated Tet proteins that contained more than the first 33, but not more than the first 97, N-terminal amino acids of Tet also increased the susceptibility to aminoglycosides and complemented the potassium uptake defects in Escherichia coli. The primary structure of this N-terminal Tet fragment has the hydropathic characteristics of a multimeric, transmembrane structure and is highly conserved in three different classes of Tet proteins.     

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.     

Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins

Phylogenetic analysis of tetracycline resistance genes encoding the ribosomal protection proteins (RPPs) revealed the monophyletic origin of these genes. The most deeply branching class, exemplified by tet and otrA, consisted of genes from the antibiotic-producing organisms Streptomyces rimosus and Streptomyces lividans. With a high degree of confidence, the corresponding genes of the other seven classes (Tet M, Tet S, Tet O, Tet W, Tet Q, Tet T, and TetB P) formed phylogenetically distinct separate clusters. Based on this phylogenetic analysis, a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources was developed and characterized. A pair of degenerate primers targeted all tetracycline resistance genes encoding RPPs except otrA and tet, and seven other primer pairs were designed to target the specific classes. The primers were used to detect the circulation of these genes in the rumina of cows, in swine feed and feces, and in swine fecal streptococci. Classes Tet O and Tet W were found in the intestinal contents of both animals, while Tet M was confined to pigs and Tet Q was confined to the rumen. The tet(O) and tet(W) genes circulating in the microbiota of the rumen and the gastrointestinal tract of pigs were identical despite the differences in animal hosts and antibiotic use regimens. Swine fecal streptococci uniformly possessed the tet(O) gene, and 22% of them also carried tet(M). This population could be considered one of the main reservoirs of these two resistance genes in the pig gastrointestinal tract. All classes of RPPs except Tet T and TetB P were found in the commercial components of swine feed. This is the first demonstration of the applicability of molecular ecology techniques to estimation of the gene pool and the flux of antibiotic resistance genes in production animals.     

Purification and characterization of Tet(M), a protein that renders ribosomes resistant to tetracycline

The tet(M) tetracycline resistance gene has been found in a wide variety of clinically important bacteria. It has been shown previously (Burdett, V. (1986) J. Bacteriol. 165, 564-569) that the tet(M) gene product mediates resistance at the level of protein synthesis as judged by in vitro assay. Using this assay, large amounts of protein were purified from an Escherichia coli overproducer expressing the gene under control of a T7 promoter. The purified activity consists of a single polypeptide of molecular weight 68,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was confirmed to be the tet(M) gene product by amino-terminal sequence analysis. Purified Tet(M) has an associated ribosome-dependent GTPase with the specific activity being similar to that of the corresponding activity associated with elongation factor G. Since Tet(M) also displays substantial homology to elongation factor G throughout its length, Tet(M) may function as an analog of this elongation factor.     

Evidence for recent intergeneric transfer of a new tetracycline resistance gene, tet(W), isolated from Butyrivibrio fibrisolvens, and the occurrence of tet(O) in ruminal bacteria

We have previously reported high-frequency transfer of tetracycline resistance between strains of the rumen anaerobic bacterium Butyrivibrio fibrisolvens . Donor strains were postulated to carry two Tc^R genes, one of which is transferred on a novel chromosomal element. It is shown here that coding sequences within the non-transmissible gene in B. fibrisolvens 1.230 are identical to those of the Streptococcus pneumoniae tet (O) gene. This provides the first evidence for genetic exchange between facultatively anaerobic bacteria and rumen obligate anaerobes. In contrast, the product of the transmissible Tc^R gene shares only 68% amino acid sequence identity with the TetO and TetM proteins and represents a new class of ribosome protection tetracycline resistance determinant, designated Tet W. The tet (W) coding region shows a higher DNA G + C content (53%) than other B. fibrisolvens genes or other ribosome protection-type tet genes, suggesting recent acquisition from a high G + C content genome. Tet (W) genes with almost identical sequences are also shown to be present in Tc^R strains of B. fibrisolvens from Australian sheep and in Tc^R strains of two other genera of rumen obligate anaerobes, Selenomonas ruminantium and Mitsuokella multiacidus . This provides compelling evidence for recent intergeneric transfer of resistance genes between ruminal bacteria. Tet (W) is not restricted to ruminal bacteria, as it was also present in a porcine strain of M. multiacidus .     

Gene duplication in the evolution of the two complementing domains of gram-negative bacterial tetracycline efflux proteins

The resistance of Gram- bacteria to the broad-spectrum antibiotic tetracycline (Tc) results from energy-dependent drug efflux mediated by the tet gene product, the cytoplasmic membrane Tet protein. Amino acid (aa) sequences deduced from total tet nucleotide sequences of three different resistance determinants (classes A, B and C) indicate that the protein products [Tet(A), Tet(B), and Tet(C)] share a common ancestor. Hydropathic analysis of Tet sequences predicts twelve transmembrane segments in each protein, with six occurring in each half of the molecule. More importantly, the linear distributions of these segments in the N- and C-terminal halves are nearly identical, suggesting that the two halves of each Tet protein are related by a process of tandem gene duplication and divergence. Indeed, a variable but significant conservation of sequence was detected among the N- and C-terminal halves for all possible comparisons of the three proteins. Such conservation was not observed within other prokaryotic integral membrane proteins or when other prokaryotic proteins were compared to Tet halves. Similarity, both in sequence and in predicted transmembrane structural organization, strongly suggests that a common ancestor of Tet(A), Tet(B), and Tet(C) arose by duplication of a gene reading frame specifying a transmembrane protein of approximately 200 aa residues. The two halves of Tet proteins correspond to the two domains, alpha and beta, which have distinct, complementary roles in Tc efflux. Nevertheless, selective constraints to function in the cytoplasmic membrane have apparently led to maintenance of similar patterns of secondary structural organization in these complementary domains.     

Identification of a new ribosomal protection type of tetracycline resistance gene,tet(36), from swine manure pits

Previously, only one ribosome protection type of a tetracycline resistance gene, tetQ, had been identified in Bacteroides spp. During an investigation of anaerobic bacteria present in swine feces and manure storage pits, a tetracycline-resistant Bacteroides strain was isolated. Subsequent analysis showed that this new Bacteroides strain, Bacteroides sp. strain 139, did not contain tetQ but contained a previously unidentified tetracycline resistance gene. Sequence analysis showed that the tetracycline resistance gene from Bacteroides sp. strain 139 encoded a protein (designated Tet 36) that defines a new class of ribosome protection types of tetracycline resistance. Tet 36 has 60% amino acid identity over 640 aa to TetQ and between 31 and 49% amino acid identity to the nine other ribosome protection types of tetracycline resistance genes. The tet(36) region was not observed to transfer from Bacteroides sp. strain 139 to another Bacteroides sp. under laboratory conditions. Yet tet(36) was found in other genera of bacteria isolated from the same swine manure pits and from swine feces. Phylogenetic analysis of the tet(36)-containing isolates indicated that tet(36) was present not only in the Cytophaga-Flavobacter-Bacteroides group to which Bacteroides sp. strain 139 belongs but also in gram-positive genera and gram-negative proteobacteria, indicating that horizontal transfer of tet(36) is occurring between these divergent phylogenetic groups in the farm environment.     

Determinants of resistance to chlortetracycline and other antibiotics in chlortetracycline-producing strain of Streptomyces aureofaciens

Data are presented on resistance of Streptomyces aureofaciens strain TB-633 FU--the producer of chlortetracycline (CTC) to autogenous antibiotics and a number of other antibiotics. It is demonstrated that resistance to CTC is specified by ctr genes of constitutive expression as well as by inducible genes. CTC and ethidium bromide may serve as efficient inductors of inducible ctr genes. The induction process is accompanied by increase in antibiotic biosynthesis level. Genes responsible for strain resistance to a number of macrolide antibiotics and thiostrepton are inducible and only function in the presence of appropriate antibiotics in the medium. The action of inducible mtr gene(s) is described in detail. The gene(s) simultaneously ensure increase in resistance to CTC and a number of macrolide antibiotics in the presence of exogenous inductors in media, such as both CTC and macrolide antibiotics. Mutants have been isolated which provide constitutive level of resistance to these antibiotics. A series of ctr and mtr mutants have increased CTC biosynthesis as compared to the initial level. Data on comparative analysis of the results obtained from hybridization of fragments of S. aureofaciens and S. rimosus DNAs to actI and actIII genes, responsible for polyketide synthases' synthesis, demonstrate that genes for CTC and OTC biosynthesis are situated on DNA fragments of similar size. This determines the strategy for cloning ctr and mtr genes as well as genes for CTC biosynthesis from S. aureofaciens.     

Overproduction and purification of the Tn10-specified inner membrane tetracycline resistance protein Tet using fusions to β-galactosidase

Tetracycline resistance in the Enterobacteriaceae is mediated by a number of genetically related, usually plasmid-borne, determinants which specify an efflux system involving an inner membrane protein, Tet. Attempts to overproduce the Tn10 (Class B)-encoded Tet in Escherichia coli by cloning the structural gene tet downstream of the lambda PL promoter under regulation by temperature-sensitive lambda repressor cI857 were unsuccessful; induction at 42 degrees C resulted in filamentous, non-viable cells containing little detectable overproduction of the protein. However, cells containing tet fused to lacZ were resistant to tetracycline at 30 degrees C and synthesized modest amounts of a large fusion protein when induced at 42 degrees C. Fusion of the N-terminal half or the first 38 amino acids of tet to lacZ did lead to increased production of fusion proteins. Fusions could be purified by size or by LacZ immunoaffinity or substrate-affinity chromatography. In the latter method, selected detergents were required to counteract nonspecific binding of Tet to the adsorbant. Amino acid sequencing of the N-terminus of Tet-LacZ fusion proteins indicated that most molecules were blocked at this terminus. The sequence of an unblocked subpopulation was consistent with that expected from the nucleotide sequence. A collagen peptide linker, genetically placed between tet and lacZ, allowed recovery of purified Tet protein after collagenase treatment of the purified fusion protein.     

Tet protein domains interact productively to mediate tetracycline resistance when present on separate polypeptides

Both domains, alpha and beta, of the cytoplasmic membrane-localized Tet proteins encoded by the tet gene family (classes A through E) are required for resistance to tetracycline (Tcr) in gram-negative bacteria. Two inactive proteins, each containing a mutation in the opposite domain, are capable of complementation to produce Tcr. Similarly, inactive hybrid proteins expressed by interdomain gene hybrids constructed between tet(B) and tet(C) [tet(B) alpha/(C) beta and tet(C) alpha/(B) beta] together produce significant Tcr via trans complementation (R.A. Rubin and S. B. Levy, J. Bacteriol. 172:2303-2312, 1990). A derivative of tet(B) was constructed to express the two domains of Tet(B) as separate polypeptides, neither containing intact the central, hydrophilic interdomain region. Cells harboring this tet(B) mutant expressed Tcr at about 20% the level conferred by intact tet(B). As expected, no detectable amount of a full-length Tet protein was expressed. A polypeptide corresponding to the alpha domain was observed. Interdomain hybrids between tet(B) and tet(C) containing a frameshift at the fusion junction, designed to result in expression of each of the four domains on separate polypeptides, showed trans complementation without production of detectable full-length proteins. Levels of Tcr were greater than or equal to those previously observed in complementations using full-length hybrid proteins. These results strongly suggest that polypeptides harboring individual alpha and beta domains, lacking an intact interdomain region, can interact productively in the cell to confer Tcr.     

Molecular cloning of chlortetracycline resistance gene from chlortetracycline producer Streptomyces aureofaciens

The chlortetracycline (CT) resistance gene ctr was cloned from S. aureofaciens 633, a strain producing the antibiotic. The 6.6-kb DNA Bam HI fragment containing the resistance gene was cloned with the plasmid vector pIJ699. Comparison of the restriction maps of the cloned gene and the oxytetracycline (OT) resistance gene otrA from S. rimosus revealed their similarity which enabled identification of the cloned resistance gene as otrA. Investigation of the resistance determinants in S. aureofaciens 633 made it possible to identify a mtr gene(s). It was demonstrated that introduction of a ctrA gene into S. lividance provided a simultaneous increase in the resistance of the recipient strain to CT and a number of macrolide antibiotics. The CT resistance determinants in S. lividans TK64 showed properties of exogenous induction by CT and the macrolide antibiotics similar to the properties of the mtr gene(s) of S. aureofaciens. Possible adaptation properties of mtr genes are discussed.     

Interdomain hybrid Tet proteins confer tetracycline resistance only when they are derived from closely related members of the tet gene family

Inner membrane Tet proteins encoded by tet genes in gram-negative bacteria mediate resistance to tetracycline (Tcr) by directing its export. Total sequences for class A, B, and C tet genes demonstrate that their products have a common ancestor, with Tet(A) and Tet(C) being more closely related (78% identical) than either is to Tet(B) (45% identical). The N- and C-terminal halves of Tet(B) and Tet(C) appear to comprise separate domains, and trans-complementation observed between tetracycline sensitive mutants in either domain of Tet(B) suggests separate but interactive functions for these domains. In this present study, interdomain hybrid genes were constructed to express hybrid tet products whose N- and C-terminal halves were derived from different family members [Tet(A/C), Tet(B/C), and Tet(C/B)]. Tet(A/C) specified a level of Tcr comparable to wild-type Tet(C) and 60% that of Tet(A), indicating that domains from these closely related tet products can function in cis. Although neither Tet(B/C) nor Tet(C/B) hybrids conferred significant Tcr, cells producing both of these types of hybrid proteins expressed substantial Tcr, indicating that productive interactions can occur in trans between Tet(B/C) and Tet(C/B). Taken together, these results suggest that highly specific interactions between the N- and C-terminal domains are necessary for Tcr and do not occur in individual hybrids derived from the more distant relatives, Tet(B) and Tet(C). This requirement for specific interactions suggests that N- and C-terminal domains have coevolved in each member of the Tet family.     

Silencing and un-silencing of tetracycline-controlled genes in neurons

To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (P(tets)). We have discovered that stably integrated P(tet) becomes functionally silenced in the majority of neurons when it is inactive during development. P(tet) silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced P(tet) gene silencing, possibly by inducing promoter accessibility.     

Presence of specific antibiotic (tet) resistance genes in infant faecal microbiota

The widespread use of antibiotics for medical and veterinary purposes has led to an increase of microbial resistance. The antibiotic resistance of pathogenic bacteria has been studied extensively. However, antibiotics are not only selective for pathogens: they also affect all members of the gut microbiota. These microorganisms may constitute a reservoir of genes carrying resistance to specific antibiotics. This study was designed to characterize the gut microbiota with regard to the presence of genes encoding tetracycline resistance proteins (tet) in the gut of healthy exclusively breast-fed infants and their mothers. For this purpose we determined the prevalence of genes encoding ribosomal protection proteins (tet M, tet W, tet O, tet S, tet T and tet B) by PCR and characterized the gut microbiota by FISH in stools of infants and their mothers. The gene tet M was found in all the breast-fed infants and their mothers. tet O was found in all of the mothers' samples, whilst only 35% of the infants harboured this gene. tet W was less frequently found (85% of the mothers and 13% of the infants). None of the other genes analysed was found in any sample. Our results suggest that genes carrying antibiotic resistance are common in the environment, as even healthy breast-fed infants with no direct or indirect previous exposure to antibiotics harbour these genes.     

Second-site suppressor mutations for the serine 202 to phenylalanine substitution within the interdomain loop of the tetracycline efflux protein Tet(C)

The serine 202 to phenylalanine substitution within the cytoplasmic interdomain loop of Tet(C) greatly reduces tetracycline resistance and efflux activity (Saraceni-Richards, C. A., and Levy, S. B. (2000) J. Biol. Chem. 275, 6101-6106). Second-site suppressor mutations were identified following hydroxylamine and nitrosoguanidine mutagenesis. Three mutations, L11F in transmembrane 1 (TM1), A213T in the central interdomain loop, and A270V in cytoplasmic loop 8-9, restored a wild type level of resistance and an active efflux activity in Escherichia coli cells bearing the mutant tet(C) gene. The Tet S202F protein with the additional A270V mutation was expressed in amounts comparable with the original mutant, whereas L11F and A213T Tet(C) protein mutants were overexpressed. Introduction of each single mutation into the wild type tet(C) gene by site-directed mutagenesis did not alter tetracycline resistance or efflux activity. These secondary mutations may restore resistance by promoting a conformational change in the protein to accommodate the S202F mutation. The data demonstrate an interaction of the interdomain loop with other distant regions of the protein and support a role of the interdomain loop in mediating tetracycline resistance.