Diane E. Taylor

Ribosomal Protection Proteins and Their Mechanism of Tetracycline Resistance

Ribosomal protection represents an important tactic for promoting tetracycline resistance in both gram-positive and -negative species. Tet(O) and Tet(M) are the best studied of these determinants and were originally isolated from Campylobacter jejuni and Streptococcus spp., respectively, although both are widely distributed (10). These are the only two ribosomal protection proteins (RPPs) that have been studied in detail, and therefore, they have been dealt with extensively in this review. It is assumed, however, that the other members of this class of RPPs [Tet(S), Tet(T), Tet(Q), TetB(P), Tet(W), and OtrA] function through similar mechanisms. The distribution of these determinants in the eubacteria has been extensively reviewed by Chopra and Roberts (10) and more recent information can also be found at http://faculty.washington.edu/marilynr/. n nAlthough this review focuses primarily on RPPs, it should be noted that a great variety of tetracycline resistance mechanisms exist (for a review, see reference 10). These determinants include (i) the efflux-based mechanisms found in gram-positive and gram-negative bacteria (10), (ii) the enzymatic degradation of tetracyclines found in Bacteroides (46), (iii) the rRNA mutations found in Propionibacterium acnes and Helicobacter pylori (19, 40, 55), and (iv) a host of undetermined mechanisms which bear little resemblance to the well-documented determinants mentioned above (10). n nIn this review, we will survey recent advances in the study of the ribosome, tetracycline, and the RPPs that further the understanding of RPP activity. Earlier work dealing with Tet(M) and Tet(O) as well as the other RPPs has been reviewed previously (51, 52).

Spontaneous Mutations That Confer Antibiotic Resistance in Helicobacter pylori

ABSTRACT In this study, we systematically examined in vitro frequencies and spectra of the spontaneous mutations in Helicobacter pylori that confer resistance to clarithromycin (Clar), metronidazole (Mtzr), amoxicillin (Amxr), ciprofloxacin (Cipr), and rifampin (Rifr). The mutation rate of Rifror Cipr determined in a fluctuation assay is 1 × 10−8 to 2 × 10−8 per cell per division. In contrast, the mutation rates of Clar, Mtzr, and Amxr are much lower (<10−9). However, Mtzr mutants could be readily selected in vitro by using the serial passage method, suggesting that the mutagenic effect and selective effect of a sublethal dose of metronidazole contribute to the rapid development of Mtzr. Analysis of spontaneous Rifr, Clar, and Cipr mutants confirmed previous results indicating that mutations within therpoB gene, the 23S rRNA gene, and thegyrA gene, respectively, are responsible; also, several new mutant alleles were identified. Mtzrmutants resulted most frequently, but not always, from mutations in the rdxA gene. DNA fragments containing each mutant allele could readily transform susceptibleH. pylori strains to resistance, confirming that each mutant allele is responsible for the resistance phenotype.

Macrolide Resistance in Campylobacter jejuni and Campylobacter coli: Molecular Mechanism and Stability of the Resistance Phenotype

ABSTRACT A collection of 23 macrolide-resistant Campylobacter isolates from different geographic areas was investigated to determine the mechanism and stability of macrolide resistance. The isolates were identified as Campylobacter jejuni or Campylobacter coli based on the results of the hippurate biochemical test in addition to five PCR-based genotypic methods. Three point mutations at two positions within the peptidyl transferase region in domain V of the 23S rRNA gene were identified. About 78% of the resistant isolates exhibited an A→G transition at Escherichia coli equivalent base 2059 of the 23S rRNA gene. The isolates possessing this mutation showed a wide range of erythromycin and clarithromycin MICs. Thus, this mutation may incur a greater probability of treatment failure in populations infected by resistant Campylobacter isolates. Another macrolide-associated mutation (A→C transversion), at E. coli equivalent base 2058, was detected in about 13% of the isolates. An A→G transition at a position cognate with E. coli 23S rRNA base 2058, which is homologous to the A2142G mutation commonly described in Helicobacter pylori, was also identified in one of the C. jejuni isolates examined. In the majority of C. jejuni isolates, the mutations in the 23S rRNA gene were homozygous except in two cases where the mutation was found in two of the three copies of the target gene. Natural transformation demonstrated the transfer of the macrolide resistance phenotype from a resistant Campylobacter isolate to a susceptible Campylobacter isolate. Growth rates of the resulting transformants containing A-2058→C or A-2059→G mutations were similar to that of the parental isolate. The erythromycin resistance of six of seven representative isolates was found to be stable after successive subculturing in the absence of erythromycin selection pressure regardless of the resistance level, the position of the mutation, or the number of the mutated copies of the target gene. One C. jejuni isolate showing an A-2058→G mutation, however, reverted to erythromycin and clarithromycin susceptibility after 55 subcultures on erythromycin-free medium. Investigation of ribosomal proteins L4 and L22 by sequence analysis in five representative isolates of C. jejuni and C. coli demonstrated no significant macrolide resistance-associated alterations in either the L4 or the L22 protein that might explain either macrolide resistance or enhancement of the resistance level.

Incidence of Antibiotic Resistance in Campylobacter jejuni Isolated in Alberta, Canada, from 1999 to 2002, with Special Reference to tet(O)-Mediated Tetracycline Resistance

ABSTRACT Of 203 human clinical isolates of Campylobacter jejuni from Alberta, Canada (1999 to 2002), 101 isolates (50%) were resistant to at least 64 μg of tetracycline/ml, with four isolates exhibiting higher levels of tetracycline resistance (512 μg/ml). In total, the MICs for 37% of tetracycline-resistant isolates (256 to 512 μg/ml) were higher than those previously reported in C. jejuni (64 to 128 μg/ml). In the tetracycline-resistant clinical isolates, 67% contained plasmids and all contained the tet(O) gene. Four isolates resistant to high levels of tetracycline (MIC = 512 μg/ml) contained plasmids carrying the tet(O) gene, which could be transferred to other isolates of C. jejuni. The tetracycline MICs for transconjugants were comparable to those of the donors. Cloning of tet(O) from the four high-level tetracycline-resistant isolates conferred an MIC of 32 μg/ml for Escherichia coli DH5α. In contrast, transfer to a strain of C. jejuni by using mobilization conferred an MIC of 128 μg/ml. DNA sequence analysis determined that the tet(O) genes encoding lower MICs (64 to 128 μg/ml) were identical to one other, although the tet(O) genes encoding a 512-μg/ml MIC demonstrated several nucleotide substitutions. The quinolone resistance determining region of four ciprofloxacin-resistant isolates (2%) was analyzed, and resistance was associated with a chromosomal mutation in the gyrA gene resulting in a Thr-86-Ile substitution. In addition, six kanamycin-resistant isolates contained large plasmids that carry the aphA-3 marker coding for 3′-aminoglycoside phosphotransferase. Resistance to erythromycin was not detected in 203 isolates. In general, resistance to most antibiotics in C. jejuni remains low, except for resistance to tetracycline, which has increased from about 8 to 50% over the past 20 years.

Studies of Temperature-sensitive Transfer and Maintenance of H Incompatibility Group Plasmids

The mechanism of temperature-sensitive transfer was studied for plasmids of the H incompatibility group. Transfer depended on the temperature of the mating mixture but the growth temperature of the donor was also important, and donor cells previously grown at 26 degrees C could not facilitate transfer at 37 degrees C. Comparison of transfer characteristics of a non-thermosensitive H plasmid R831b and thermosensitive H plasmids from Salmonella from Ontario during a 2 h mating period showed that the thermosensitive phenotype inhibited transfer by about 200-fold at 37 degrees C and by 10-fold at 42 degrees C. At temperatures between 15 and 30 degrees C, the thermotolerant H plasmid transferred at about the same frequency as the temperature-sensitive plasmid. Elimination of some H plasmids after growth of host cells was also observed and physical evidence of this was obtained. The characteristic of high-temperature elimination (Hte) was limited to plasmids from similar bacterial and geographphical sources. Plasmids from Salmonella spp. isolated in Ontario did not possess this phenotype, whereas plasmids from Serratia marcescens isolated in the United States did. Although the Tra(ts) and Hte phenotypes may both be characteristic of H plasmids, they were shown to be separate and distinct properties. The H plasmids used in this study were isolated and their molecular weights determined by agarose gel electrophoresis. All were large, with molecular weights often exceeding 140 X 10(6). In contrast, the thermostable H plasmid R831b had a molecular weight on only 49 X 10(6).

Campylobacter jejuni Drives MyD88-Independent Interleukin-6 Secretion via Toll-Like Receptor 2

ABSTRACT Gastrointestinal disease caused by Campylobacter jejuni is characterized by localized inflammation and the destruction of the epithelial cell barrier that forms host innate protection against pathogens. This can lead to an imbalance in fluid transport across the gastrointestinal tract, resulting in severe diarrhea. The mechanisms of host cell receptor recognition of C. jejuni and downstream immune signaling pathways leading to this inflammatory disease, however, remain unclear. The aim of this study was to analyze the mechanisms involved in C. jejuni induction of the acute-phase inflammatory response regulator interleukin-6 (IL-6). Polarized intestinal epithelial Caco-2 monolayers responded to infections with Salmonella enterica serovar Typhimurium and eight isolates of C. jejuni by an increase in levels of expression and secretion of IL-6. No such IL-6 response, however, was produced upon infection with the human commensal organism Lactobacillus rhamnosus GG. The IL-6 signaling pathway was further characterized using short interfering RNA complexes to block gene expression. The inhibition of myeloid differentiation primary response protein 88 (MyD88) expression in this manner did not affect C. jejuni-induced IL-6 secretion, suggesting a MyD88-independent route to IL-6 signal transduction in C. jejuni-infected human epithelial cells. However, a significant reduction in levels of IL-6 was evident in the absence of Toll-like receptor 2 (TLR-2) expression, implying a requirement for TLR-2 in C. jejuni recognition. Caco-2 cells were also treated with heat-inactivated and purified membrane components of C. jejuni to isolate the factor responsible for triggering IL-6 signaling. The results demonstrate that C. jejuni surface polysaccharides induce IL-6 secretion from intestinal epithelial cells via TLR-2 in a MyD88-independent manner.

The tellurite-resistance determinants tehAtehB and klaAklaBtelB have different biochemical requirements.

The tehAtehB operon from the Escherichia coli chromosome (32.3 min) mediates resistance to potassium tellurite (K2TeO3) when expressed on a multicopy plasmid such as pUC8 (pTWT100). An MIC of 128 micrograms ml-1 is observed when tehAtehB is expressed in a wild-type host and grown on rich media. In this study, the tehAtehB determinant was transformed into mutants deficient in electron transport processes and/or thiol redox coupling within E. coli. These mutants included ubi, nad, cys, nar, trx, grx, gsh and sod. MICs of tehAtehB transformed into these mutants ranged from 1-16 micrograms K2TeO3 ml-1 compared to 0.03-2 micrograms ml-1 for strains transformed with a control plasmid. The tellurite-resistance determinant locus kilA cloned from the IncP alpha plasmid RK2Ter (pDT1558) was also investigated in these strains. This tellurite-resistance determinant showed little or no dependency on the host genotype. The ability of tehAtehB to mediate resistance in wild-type hosts is limited to rich medium. Rich medium may provide a key unidentified cofactor required by TehATehB that is not provided under minimal conditions. Again, the ability of the kilA determinant to mediate tellurite resistance was independent of medium conditions. These data suggest that either a reducing environment or electron-reducing equivalents are required for tehAtehB to mediate high levels of resistance to potassium tellurite. Therefore, the two resistance determinants studied here possess two very different biochemical mechanisms of resistance. Our data also suggest a mechanism for endogenous resistance to tellurite which involves nitrate reductase, superoxide dismutase, and thiol redox processes.

pVir and Bloody Diarrhea in Campylobacter jejuniEnteritis

The plasmid pVir may play a role in the virulence of Campylobacter jejuni, a leading cause of bacterial gastroenteritis. The pVir plasmid was identified in 17% of 104 C. jejuni clinical isolates studied and was significantly associated with the occurrence of blood in patient stool, a marker of invasive infection. The pVir plasmid was not associated with greater occurrence of diarrhea, fever, pain, vomiting, or need for patient hospitalization. Isolates containing pVir were also associated with the presence of a tetracycline-resistance plasmid, but pVir did not transfer with tetracycline-resistance plasmids to recipient strains of C. jejuni. The association of pVir and bloody stool suggests that pVir may be clinically relevant in C. jejuni infections.

16S rRNA Mutations That Confer Tetracycline Resistance in Helicobacter pylori Decrease Drug Binding in Escherichia coli Ribosomes

Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.

Characterization of Plasmid-Mediated aphA-3 Kanamycin Resistance in Campylobacter jejuni

A total of 254 isolates of Campylobacter jejuni and three isolates of Campylobacter coli, isolated from Sweden, Canada, and Egypt, were screened for kanamycin resistance. Eight strains of C. jejuni contained large plasmids that carried the aphA-3 kanamycin-resistance marker. In six plasmids, the aphA-3 gene was located downstream of an apparent insertion sequence, designated IS607*, which showed a considerable similarity to IS607, characterized on the chromosome of some Helicobacter pylori strains. In contrast, the other plasmids carried the aphA-3 gene as a part of a resistance cluster. This included three resistance markers encoding 6-adenylyltransferase (aadE), streptothricin acetyltransferase (sat), and 3-aminoglycoside phosphotransferase type III (aphA-3). The genetic organization of this resistance cluster suggests that it has been acquired by C. jejuni from a Gram-positive organism. The IS607* element was also observed in kanamycin-susceptible strains of C. jejuni on plasmids mediating tetracycline resistance. The kanamycin-resistance phenotype transferred along with tetracycline resistance by conjugation from four representative C. jejuni strains to a recipient strain of C. jejuni. The kanamycin-resistance determinant (aphA-3) was stably transferred from one of the four C. jejuni strains to a recipient strain of Escherichia coli. However, the C. jejuni plasmid, which also carries the tetO gene, was not maintained in E. coli. Pulsed-field gel electrophoresis revealed the integration of approximately 50 kb of the plasmid into the chromosome of the E. coli recipient.